Differentiation of non-insulin producing cells into insulin producing cells by GLP-1 or exendin-4 and uses thereof

ABSTRACT

The present invention relates to a population of insulin producing cells made by a process comprising contacting non-insulin producing cells with a growth factor selected from the group consisting of GLP-1 or Exendin-4, growth factors having amino acid sequences substantially homologous to GLP-1 or Exendin-4, and fragmets thereof. The present invention also relates to methods of differentiating non-insulin producing cells into insulin producing cells and of enriching a population of cells for insulin-producing cells. The present invention also relates to methods of treating diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/095,917, filed Aug. 10, 1998.

The government has certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a population of insulin producing cellsdifferentiated from non-insulin producing cells by contacting thenon-insulin producing cells with Glucagon-like peptide-1 (“GLP-1”),exendin-4, or related peptides. The present invention also relates tothe methods for obtaining the insulin producing cells and therapeuticuses in the treatment of diabetes mellitus.

2. Background Art

The mammalian pancreas is composed of two distinct types of glandulartissue, the exocrine cells that secrete digestive enzymes into theintestine and the endocrine cells that secrete hormones into the bloodstream. Endocrine cells were traditionally believed to develop from theneural crest whereas the exocrine cells were believed to develop fromthe endoderm. More recent work suggests that these two cell types cancome from common endodermal precursor cells located along the epitheliallining of the ducts (Teitelman, 1996). It should be noted that theendocrine cells are terminally differentiated and do not divide to makenew endocrine cells. Pancreatic endodermal precursor cells are the onlycells thought to produce new pancreatic endocrine cells.

The pancreas consists of ducts, which carry the exocrine enzymes(amylase and lipase) to the intestine; acinar cells, which produce theexocrine enzymes; and islets of Langerhans, which contain the endocrinecells that produce and secrete insulin, amylin, and glucagon. Thesehormones help to maintain normal blood glucose levels within aremarkably narrow range.

Among the islet cells are beta cells which produce and secrete insulin.Insulin production and secretion by the beta cells is controlled byblood glucose levels. Insulin release increases as blood glucose levelsincrease. Insulin promotes the uptake of glucose by target tissues and,thus, prevents hyperglycemia by shuttling glucose into tissues forstorage.

Beta cell dysfunction and the concomitant decrease in insulin productioncan result in diabetes mellitus. In Type 1 diabetes, the beta cells arecompletely destroyed by the immune system, resulting in an absence ofinsulin producing cells (Physician's Guide to Insulin Dependent [Type I]Diabetes Mellitus: Diagnosis and Treatment, American DiabetesAssociation, 1988). In Type 2 diabetes, the beta cells becomeprogressively less efficient as the target tissues become resistant tothe effects of insulin on glucose uptake. Type 2 diabetes is aprogressive disease and beta cell function continues to deterioratedespite on-going treatment with any presently available agent (UKProspective Study Group, 1995). Thus, beta cells are absent in peoplewith Type 1 diabetes and are functionally impaired in people with Type 2diabetes.

Beta cell dysfunction currently is treated in several different ways. Inthe treatment of Type 1 diabetes or the late stages of Type 2 diabetes,insulin replacement therapy is used. Insulin therapy, althoughlife-saving, does not restore normoglycemia, even when continuousinfusions or multiple injections are used in complex regimes. Forexample, postprandial levels of glucose continue to be excessively highin individuals on insulin replacement therapy. Thus, insulin therapymust be delivered by multiple daily injections or continuous infusionand the effects must be carefully monitored to avoid hyperglycemia,hypoglycemia, metabolic acidosis, and ketosis.

Replacement of beta cells can be achieved with pancreatic transplants.(Scharp et al., 1991; Warnock et al., 1991). Such transplants, however,require finding a matching donor, surgical procedures for implanting theharvested tissue, and graft acceptance. After transplantation in aperson with Type 1 diabetes, on-going immunosuppression therapy isrequired because cell surface antigens on the beta cells are recognizedand attacked by the same processes that destroyed the beta cellsoriginally. Immunosuppressive drugs, such as cyclosporin A, however,have numerous side-effects, including the increase in potential forinfection. Transplantation, therefore, can result in numerouscomplications.

People with Type 2 diabetes are generally treated with drugs thatstimulate insulin production and secretion from the beta cells. A majordisadvantage of these drugs, however, is that insulin production andsecretion is promoted regardless of the level of blood glucose. Thus,food intake must be balanced against the promotion of insulin productionand secretion to avoid hypoglycemia or hyperglycemia.

In recent years several new agents have become available to treat Type 2diabetes. These include metformin, acarbose and troglitazone (seeBressler and Johnson, 1997). However, the drop in hemoglobin A1cobtained by these newer agents is less than adequate (Ghazzi et al.,1997), suggesting that they will not improve the long-term control ofdiabetes mellitus.

Most recently, glucagon-like peptide-1 (GLP-1), a hormone normallysecreted by neuroendocrine cells of the gut in response to food, hasbeen suggested as a new treatment for Type 2 diabetes (Gutniak et al.,1992; Nauck et al., J. Clin. Invest., 1993). It increases insulinrelease by the beta cells even in subjects with long-standing Type 2diabetes (Nauck et al., Diabetologia, 1993). GLP-1 treatment has anadvantage over insulin therapy because GLP-1 stimulates endogenousinsulin secretion, which turns off when blood glucose levels drop (Naucket al., Diabetologia, 1993; Elahi et al., 1994), when blood glucoselevels are high. GLP-1 promotes euglycemia by increasing insulin releaseand synthesis, inhibiting glucagon release, and decreasing gastricemptying (Nauck et al., Diabetologia, 1993; Elahi et al., 1994; Wills etal., 1996; Nathan et al., 1992; De Ore et al., 1997). GLP-1 also inducesan increase in hexokinase messenger RNA levels (Wang et al.,Endocrinology 1995; Wang et al., 1996). GLP-1 is known to have a potentinsulin-secreting effect on beta cells (Thorens and Waeber, 1993;Orskov, 1992) and to increase insulin biosynthesis and proinsulin geneexpression when added to insulin-secreting cell lines for 24 hours(Drucker et al., 1987; Fehmann and Habener, 1992). In studies using RIN1046-38 cells, twenty-four hour treatment with GLP-1 increased glucoseresponsiveness even after the GLP-1 had been removed for an hour andafter several washings of the cells (Montrose-Rafizadeh et al., 1994).Thus, GLP-1 is an insulin releasing agent and an insulinotropic agent(i.e., an agent that increases insulin synthesis) known to have aprolonged effect on beta cells. GLP-1 is a product of posttranlationalmodification of proglucagon. The sequences of GLP-1 and its activefragments GLP-1 (7–37) and GLP-1 (7–36) amide are known in the art(Fehmann et al., 1995).

GLP-1 receptors have been shown to be present in the gut and in thepancreatic islets (Id.). The receptors belong to a family ofG-protein-linked receptors that includes glucagon, secretin, andvasoactive intestinal peptide receptors. After binding of GLP-1 to itsreceptor there is a rise in cAMP in beta cells of the islets ofLangerhans (Widmann et al., 1996), indicating that the receptor iscoupled to the adenyl cyclase system by a stimulator G-protein. Inperipheral tissues, such as liver, fat and skeletal muscle, however, noincrease in cAMP with GLP-1 is seen, suggesting that GLP-1 acts througha different system on peripheral tissues (Valverde andVillanueva-Penacarrillo, 1996).

Exendin-4 is a peptide produced in the salivary glands of the GilaMonster lizard (Goke et al., 1993). The amino acid sequence forExendin-4 is known in the art (Fehmann et al. 1995). Although it is theproduct of a uniquely non-mammalian gene and appears to be expressedonly in the salivary gland (Chen and Drucker, 1997), Exendin-4 shares a52% amino acid sequence homology with GLP-1 and in mammals interactswith the GLP-1 receptor (Goke et al., 1993; Thorens et al., 1993). Invitro, Exendin-4 has been shown to promote insulin secretion by insulinproducing cells and, given in equimolar quantities, is more potent thanGLP-1 at causing insulin release from insulin producing cells.

In vivo studies using GLP-1 have been limited to the use of single orrepeated bolus injections or short-term infusions of GLP-1 andsubsequent evaluation of the insulin secreting effects. In one suchstudy, infusions of GLP-1 for two hours were tested in patients withType 1 diabetes for the ability of GLP-1 to promote glucose uptake inmuscle and release of glucose from the liver (Gutniak et al., 1992).Therapeutic uses of GLP-1 for increasing the release of insulin havebeen considered for Type 2 diabetes, but not for Type 1 diabetes, sinceType 1 diabetes is marked by an absence of beta cells, the known targetcell for GLP-1. Furthermore, GLP-1 has known limitations as atherapeutic agent in the treatment of diabetes because it has a shortbiological half-life (De Ore et al., 1997), even when given by a bolussubcutaneously (Ritzel et al., 1995). Exendin-4 has not been usedpreviously in in vivo studies. Thus, studies to date have neversuggested that either GLP-1 or Exendin-4 is therapeutically effective onpancreatic function in people with Type 1 diabetes or that there areGLP-1 or Exendin-4 target cells in the pancreas other than the betacells.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome or reduce the abovestated problems with the prior art by providing a population of insulinproducing cells made by a process comprising contacting non-insulinproducing cells with a growth factor selected from the group consistingof GLP-1 or Exendin-4, growth factors having amino acid sequencessubstantially homologous to GLP-1 or Exendin-4, and fragments thereof.In addition, a method of differentiating non-insulin producing cellsinto insulin producing cells, comprising contacting the non-insulinproducing cells with a growth factor selected from the group consistingof GLP-1 or Exendin-4, growth factors having amino acid sequencessubstantially homologous to GLP-1 or Exendin-4, and fragments thereof isprovided. Further provided is a method of enriching a population ofcells for insulin-producing cells, comprising contacting the populationof cells with a growth factor that promotes differentiation ofnon-insulin producing cells into insulin-producing cells.

Also provided is a method of treating diabetes in a subject diagnosedwith Type 1 diabetes, comprising administering to the subject a growthfactor selected from the group consisting of GLP-1 or Exendin-4, growthfactors having amino acid sequences substantially homologous to GLP-1 orExendin-4, and fragments thereof by continuous infusion for at leasttwenty-four hours. The present invention further overcomes the prior artby providing a method of treating diabetes in a subject, comprisingobtaining non-insulin producing cells from the subject being treated orfrom a donor; contacting the non-insulin producing cells with a growthfactor, thereby promoting differentiation of non-insulin producing cellsinto insulin-producing cells; and administering the insulin-producingcells that were promoted to differentiate from non-insulin producingcells to the diabetic subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasma insulin levels in 3 month and 22 month old animals.GLP-1 (0.2 nmol/kg) was given intravenously to fasted, anesthetizedanimals.

FIG. 2 shows plasma glucose levels during a glucose tolerance in 22month old animals. GLP-1-treated animals received 1.5 pmol/kg/min for 48hrs by subcutaneous infusion. Controls were infused with saline. Glucose(1 g/kg) was given ip and the blood glucose measured at the timesindicated. The results are a mean (±SEM) of 6 treated and 6 controlanimals. Repeated measures analysis of variance from 0–30 min showed avalue of p<0.05. Asterisks indicate: * p<0.05, **p<0.01, as determinedby unpaired Student's t test.

FIG. 3 shows plasma insulin levels during a glucose tolerance test in 22month old animals. GLP-1-treated animals received 1.5 pmol/kg/min for 48hrs by subcutaneous injection. Controls were infused with saline.Glucose (1 g/kg) was given ip and serum insulin measured at the timesindicated. The results are mean (±SEM) of 6 treated and 6 controlanimals. Repeated measures analysis of variance from 0–30 min showed avalue of p<0.05. The asterisk indicates a p<0.01, as determined byunpaired Student's t test.

FIG. 4 shows the fold increase in islet insulin content after 48 hrsinfusion of saline (control, 7 animals) or GLP-1 (1.5 pmol/kg/min, 7animals) in 22 month old rats. **p<0.01 by unpaired Student's t test.

FIG. 5 shows insulin mRNA levels in pancreata from control, GLP-1-,Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample representsan individual pancreas, with four animals in each treatment group.

FIG. 6 shows GLUT2 mRNA levels in pancreata from control, GLP-1-,Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample representsan individual pancreas, with four animals in each treatment group.

FIG. 7 shows glucokinase mRNA in pancreata from control, GLP-1-,Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample representsan individual pancreas, with four animals in each treatment group.

FIG. 8 shows plasma insulin concentrations in fasted anesthetized ratsafter intravenous (iv) boli of GLP-1 (0.4 nmol/kg) and exendin-4 (0.4nmol/kg) in 100 μl NaCl. NaCl (100 μl) was also given iv to a controlgroup. Values are expressed as mean±SEM (n=6 per group).

FIG. 9 shows insulin concentrations in fasted anaesthetized rats 2 minafter intravenous boli of exendin-4 at the concentrations shown. Valuesare expressed as mean±SEM (n=6 per exendin-4 concentration).

FIG. 10 shows the effects of GLP-1 (1 nM) and exendin-4 (1 nM) treatmentfor 1 hr on intracellular cAMP levels in islets of Langerhans. Given aremeans±SEM of 4 experiments, each done in triplicate. Exendin-4 was moreeffective than GLP-1 (p<0.01). Note that after washing some of theislets in buffer following the hr in the presence of the peptides andthen removing the islets approximately 15 min later cAMP levels hadreturned to baseline.

FIG. 11 shows a photograph of cages housing the diabetic mice takenafter nine weeks of treatment with exendin-4 (24 nmol/kg) or NaClintraperitoneally daily, 24 hours after the previous bedding change.Diabetic mice were housed two per cage. Bedding was changed every 24hours for the diabetic animals after the first few weeks of treatment.The cage on the right contained the exendin-4-treated animals while thecage on the left contained the NaCl-treated animals.

FIG. 12 shows hemoglobin A1c levels in the diabetic and non-diabeticmice given either exendin-4 (24 mmol/kg) or normal salineintraperitoneally daily for 12–13 weeks. Values are expressed asmean±SEM (n=9–10 per group).

FIG. 13 shows fasting glucose and insulin concentrations in the diabeticand non-diabetic mice given either exendin-4 (24 nmol/kg) or normalsaline intraperitoneally daily for 12–13 weeks. Values are expressed asmean±SEM (n=9–10 per group).

FIG. 14 shows CCK concentration-response curve of amylase release fromAR42J cells. Cells were treated with CCK at the concentrations shown for50 min. Amylase values are expressed as a percentage of the releasedamylase into the medium over the total amylase activity of the cells.Results are the mean±SEM of 15 experiments.

FIG. 15 shows the effects of glucagon (10 nM), GLP-1 (10 nM), andinsulin (100 nM)±CCK (1 nM), on amylase release from AR42J cells.Dexamethasone-induced AR42J cells were incubated for 50 min in presenceof the hormones. Amylase values are expressed as a percentage of thereleased amylase into the medium over the total amylase activity of thecells. Results are mean±SEM of 20 experiments, * p<0.05, **p<0.01,treatment vs. no treatment. a=p<0.01.

FIG. 16 shows the effect of 50 min of treatment with 8-Bromo-cAMP (100nM) on amylase release from AR42J cells. Amylase values are expressed asa percentage of the released amylase into the medium over the totalamylase activity of the cells. Results are mean±SEM of 3 experiments, *p<0.05, treatment vs. no treatment.

FIG. 17 shows the effects of ryanodine (RY) and thapsigargin (TG) in thepresence or absence of CCK on amylase release from AR42J cells. Amylasevalues are expressed as a percentage of the released amylase into themedium over the total amylase activity of the cells. RY and TG wereadded 30 min prior to addition of CCK, which was then added for 50 min.Results are mean±SEM of 3 experiments, **p<0.01.

FIG. 18 shows the time course of the actions of vanadate (1 mM) (Δ) andgenestein (300 μM) (●) on CCK (1 nM)-mediated amylase release from AR42Jcells. Amylase release from CCK-treated (1 nM) (∘) cells or control (notreatment) (□) cells is also shown. Amylase values are expressed as apercentage of the released amylase into the medium over the totalamylase activity of the cells. Results are the mean±SEM of 4experiments. * p<0.05, **p<0.01, vanadate or genestein plus CCKtreatment vs. CCK treatment alone.

FIG. 19 shows the effects of CCK on intracellular free [Ca²⁺]_(i) insingle AR42J cells. The bar indicates the time of exposure to 10 nM CCKin three different cells. FIG. 19A shows the typical [Ca²⁺]_(i) responseobserved in at least 85% of the cells. FIG. 19B shows that the[Ca²⁺]_(i) response to CCK is almost completely abolished following 60min exposure to 10 μM ryanodine (RY) and 500 nM thapsigargin (TG). FIG.19C shows that the [Ca²⁺]_(i) transient is abbreviated by reduction ofextracellular Ca²⁺ during exposure to CCK.

FIG. 20 shows the effects of GLP-1 on [Ca²⁺]_(i) and CCK-induced[Ca²⁺]_(i) transients in single AR42J cells. The same cell was studiedin A–C. FIG. 20A shows that exposure to 1 nM GLP-1 induced small, slow,prolonged [Ca²⁺]_(i) transients in approximately 50% of AR42J cells. Thereduction in the amplitude of the subsequent exposure to 10 nM CCK isshown in FIG. 20B. FIG. 20C shows that the amplitude of the [Ca²⁺]_(i)transient is further reduced in response to a second exposure to CCKapplied in <10 min.

FIG. 21 shows the effects of glucagon and 8-bromo-cAMP (8BcAMP) on[Ca²⁺]_(i) in single AR42J cells. FIG. 21A shows that glucagon (10 nM)induced slow, small, prolonged [Ca²⁺]_(i) transients in approximately70% of cells. FIG. 21B shows that in cells treated with 10 nM glucagonfor 3–10 min, the subsequent [Ca²⁺]_(i) transients induced by 10 nM CCKshowed a slow rate of rise as well as a prolonged relaxation phase. FIG.21C shows that brief (1–5 min) exposures to 100 nM 8BcAMP attenuated therelaxation of CCK-induced [Ca²⁺]_(i) transients.

FIG. 22 shows the effects of GLP-1 (10 nM), glucagon (10 nM) and CCK (1nM) treatment±IBMX (100 nM) for 50 min on intracellular cAMP levels inAR42J cells. Results are mean±SEM of 3 experiments, *p<0.05.

FIG. 23 shows RT-PCR of GLP-1 receptors in AR42J cells and rat pancreas.cDNA was amplified for 30 cycles using primers in the 5′-and 3′-end ofthe rat pancreatic GLP-1 receptor. PCR products were resolved on a 1%agarose gel and visualized using ethidium bromide. From left to right;Lane 1, DNA marker; Lane 2, blank; Lane 3, AR42J cells; Lane 4, ratpancreas; Lane 5, water control. In Lanes 3 and 4 we see the expected928 bp band, corresponding to the GLP-1 receptor.

FIG. 24 shows western blot analysis of GLP-1 receptor expression inAR42J (Lane 1,2) and RIN 1046-38 (Lane 3,4) cells. Cells weresolubilized and GLP-1 receptors were detected after immunoprecipitationand Western blotting with antibody to the amino-terminus of the GLP-1receptor. The positions of the molecular markers, in kDa, are on theright. The 65 and 46 kDa bands have been shown to correspond to themature and core-glycosylated GLP-1 receptors, respectively (28).

FIG. 25 shows protein tyrosine phosphorylation in AR42J cells inresponse to various stimuli. A representative anti-phosphotyrosineimmunoblot of total cellular proteins from un-treated (control) cellsand 5 min-treated cells as indicated (n=3). Note the increase intyrosine phosphorylation with CCK and sodium fluoride (NaF) of 46, 66,120 and 190 kDa bands. GLP-1 did not have any effect on those proteins.Insulin caused increased phosphorylation of 97 kDa band, correspondingto the insulin receptor β-subunit.

FIG. 26 shows immunocytochemistry of AR42J cells. AR42J cells were fixedwith glutaraldehyde, and incubated with anti-insulin or anti-glucagonantibody from Linco at a dilution of 1:300. FIG. 26A shows control AR42Jcells, anti-insulin antibody. FIG. 26B shows GLP-1 (10 nM)-treated cellsfor 48 hours, anti-insulin antibody. FIG. 26C shows GLP-1 (10nM)-treated AR42J cells for 72 hours, anti-insulin antibody. FIG. 26Dshows RIN 1046-38 insulinoma cells, anti-insulin antibody. FIG. 26Eshows control cells, anti-glucagon antibody. FIG. 26F shows GLP-1 (10nM)-treated cells for 48 hours, anti-glucagon antibody.

FIG. 27 shows the effect of time on the induction of glucagon andinsulin by GLP-1 (10 nM) production in AR42Jcells. For this experiment,cells were plated on the coverslips as described herein, all on the sameday. They were then stained with anti-insulin or anti-glucagon antibodyon the days indicated. This has now been repeated numerous times (atleast 5 times) on different days and insulin and glucagon have alwaysbeen present.

FIG. 28 shows expression of mRNAs for insulin and glucagon using RT-PCR.FIG. 28A shows insulin mRNA at 187 bp. FIG. 28B shows glucagon mRNA at236 bp. GLP-1 (1 nM) treatment was for 3 days.

FIG. 29 shows the effect of GLP-1 and exendin-4 in the presence orabsence of a protein kinase C inhibitor in AR42J cells from onerepresentative experiment, which was repeated 3 times. FIG. 29A is theautoradiogram and FIG. 29B represents the densitometry readings(relative units). Cells were plated at a density of 10⁵/well in 60 mmdishes, lysed and clarified lysates were then immunoprecipitated withanti-ERK antibody. The immune pellets were analyzed for ERK activity asdescribed in herein. Lane 1, Control AR42J cells. Lane 2, GLP-1 (10nM)-treated AR42J cells for 3 days. Lane 3, exendin-4 (0.1 nM)-treatedcells for 3 days. Lane 4, GLP-1 (10 nM)-plus exendin-4 (0.1 nM)-treatedcells for 3 days. Lane 5, GLP-1 (10 nM)-plus exendin-4-plus PKI (300μM)-treated cells for 3 days. Lane 6, exendin-4 (0.1)-plus PKI (300μM)-treated cells for 3 days. Lane 7, GLP-1 (10 nM)-plus PKI (300μM)-treated cells for 3 days. Note that exendin-4 (0.1 nM) isapproximately equivalent to GLP-1 (10 nM).

FIG. 30 shows the dose-response effect of GLP-1 on amylase release fromdexamethasone-treated AR42J cell. After 3 days treatment with differentconcentration of GLP-1 the AR42J cells were washed and 1 nM CCK wasadded. The cells were incubated for another 50 min and the samples werecollected for amylase assay. N=4, Mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

As used in the claims, “a” can mean one or more.

The present invention provides a population of insulin producing cellsmade by a process comprising contacting non-insulin producing cells witha growth factor selected from the group consisting of GLP-1 orExendin-4, growth factors having amino acid sequences substantiallyhomologous thereto, and fragments thereof. Non-insulin producing cells,including primary acinar cells, acinar cell lines (e.g., AR42J), andstem cells, that were not previously thought to have GLP-1 receptors andnot previously thought to be capable of producing insulin can respond toGLP-1 and Exendin-4, growth factors having amino acid sequencessubstantially homologous thereto, and fragments thereof bydifferentiating into insulin producing cells. The effect is to increasethe number of insulin-producing cells, an effect that is desirable inthe treatment of diabetes mellitus.

As used herein, “insulin producing cells” includes cells that synthesize(i.e., transcribe the insulin gene, translate the proinsulin mRNA, andmodify the proinsulin mRNA into the insulin protein), express (i.e.,manifest the phenotypic trait carried by the insulin gene), or secrete(release insulin into the extracellular space) insulin in a constitutiveor inducible manner. Examples of known insulin producing cells includebeta cells, which are located in the pancreatic islets in vivo. In orderto secrete insulin, an insulin producing cell also must express IDX-1.

A population of insulin producing cells made by the present inventionmay contain cells (e.g., beta cells) that produce insulin without theuse of the present methods and other cell types. The novelty of thepresent composition and methods is not negated by the presence of cellsin the population that produce insulin naturally (e.g., beta cells). Itis also contemplated that the population of insulin producing cells mayalso contain non-insulin producing cells.

By “non-insulin producing cells” is meant any cell that does notnaturally synthesize, express, or secrete insulin constitutively orinducibly. Thus, the term “non-insulin producing cells” as used hereinexcludes beta cells. Examples of non-insulin producing cells that can beused in the methods of the present invention include pancreatic non-betacells, such as amylase producing cells, acinar cells, cells of ductaladenocarcinoma cell lines (e.g., CD18, CD11, and Capan-I cells (seeBusik et al., 1997; Schaffert et al. 1997), and stem cells.Non-pancreatic cells could also be used, for example, non-pancreaticstem cells and cells of other endocrine or exocrine organs, including,for example, pituitary cells. The non-insulin producing cells can bemammalian cells or, even more specifically, human cells. Examples of thepresent method using mammalian pancreatic non-islet, pancreatic amylaseproducing cells, pancreatic acinar cells, and stem cells are providedherein. Stem cells can include pancreatic stem cells and non-pancreaticstem cells that have been promoted to produce IDX-1, Beta 2/NeuroD, andE47. Pancreatic stem cells include duct epithelial precursor cells whichgive rise to both islet and acinar cells.

The non-insulin producing cells must have GLP-1 receptors or receptorssubstantially similar to GLP-1 receptors in order to differentiate intoinsulin producing cells. Preferably, the non-insulin producing cellcould also show, upon contact with the growth factor, an increase inintracellular calcium and ERK/MAPK activity and activation of PKC.

As used in the present application, “growth factor” means a substancethat can differentiate a non-insulin producing cell into an insulinproducing cell. Preferably the growth factor will be one of a group ofinsulinotropic growth factors, including, for example, GLP-1, exendin-4,betacellulin, Hepatocyte Scatter Factor (HSF) and activin-A, orcombinations thereof, excluding betacellulin and activin-A used togetherand excluding HSF and Activin-A used together. Preferably, greater than10% of the non-insulin producing cells will differentiate into insulinproducing cells upon contact with the growth factor; and, morepreferably, at least about 20%, 30%, 40%, 50%, or more willdifferentiate into insulin producing cells upon contact with the growthfactor. Thus, a population of insulin producing cells made in vitroaccording to the present method can comprise as low as 11% and up to100% insulin producing cells.

By “amino acid sequences substantially homologous” to GLP-1 or exendin-4is meant polypeptides that include one or more additional amino acids,deletions of amino acids, or substitutions in the amino acid sequence ofGLP-1 or exendin-4 without appreciable loss of functional activity ascompared to GLP-1 or exendin-4 in terms of the ability to differentiateinsulin producing cells from non-insulin producing cells. For example,the deletion can consist of amino acids that are not essential to thepresently defined differentiating activity and the substitution(s) canbe conservative (i.e., basic, hydrophilic, or hydrophobic amino acidssubstituted for the same). Thus, it is understood that, where desired,modifications and changes may be made in the amino acid sequence ofGLP-1 and Exendin-4, and a protein having like characteristics stillobtained. It is thus contemplated that various changes may be made inthe amino acid sequence of the GLP-1 or Exendin-4 amino acid sequence(or underlying nucleic acid sequence) without appreciable loss ofbiological utility or activity and possibly with an increase in suchutility or activity.

The term “fragments,” as used herein regarding GLP-1, Exendin-4, orgrowth factors having amino acid sequences substantially homologousthereto means a polypeptide sequence of at least 5 contiguous aminoacids of either GLP-1, Exendin 4, or growth factors having amino acidsequences substantially homologous thereto, wherein the polypeptidesequence has the differentiating function of GLP-1 and Exendin-4 asdescribed herein. The present fragment may have additional functionsthat can include antigenicity, binding to GLP-1 receptors, DNA binding(as in transcription factors), RNA binding (as in regulating RNAstability or degradation). Active fragments of GLP-1 can include, forexample, GLP-1 (7–36) amide (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ IDNO:1)); GLP-1 (7–37) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:2));GLP-1 (7–35) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKG (SEQ ID NO: 3)); GLP-1(7–34) (HAEGTFTSDVSSYLEGQAAKEFIAWLVK (SEQ ID NO:4)); GLP-1 (7–33)(HAEGTFTSDVSSYLEGQAAKEFIAWLV (SEQ ID NO:5)); GLP-1 (7–32)(HAEGTFTSDVSSYLEGQAAKEFIAWL SEQ ID NO:6)); GLP-1 (7–31)(HAEGTFTSDVSSYLEGQAAKEFIAW SEQ ID NO:7)); and GLP-1 (7–30)(HAEGTFTSDVSSYLEGQAAKEFIA SEQ ID NO:8)). Active fragments of Exendin-4can include, for example, Exendin-4 (1–39)(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:9)); Exendin-4(1–38) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPP (SEQ ID NO: 10));Exendin-4 (1–37) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPP (SEQ ID NO:11)); Exendin-4 (1–36) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAP (SEQ ID NO:12)); Exendin-4 (1–35) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA (SEQ ID NO:13)); Exendin-4 (1–34) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSG (SEQ IDNO:14)); Exendin-4 (1–33) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSS (SEQ ID NO:15)); Exendin-4 (1–32) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPS (SEQ ID NO:16)); Exendin-4 (1–31) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP (SEQ ID NO:17)); and Exendin-4 (1–30) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG (SEQ IDNO:18)).

Other fragments and modified sequences of GLP-1 are known in the art(U.S. Pat. No. 5,614,492; U.S. Pat. No. 5,545,618; European PatentApplication, Publication No. EP 0658568 A1; WO 93/25579). Similarfragments and modified sequences of exendin-4 can be easilyextrapolated. It is expected that (a) the following five residues ofGLP-1, an H residue at position 7; a G residue at position 10; an Fresidue at position 12; a T residue at position 13 and a D residue atposition 15, and (b) the following five residues of exendin-4, an Hresidue at position 1; a G residue at position 4, an F residue atposition 6; a T residue at position 7 and a D residue at position 9,should be included in a fragment since these residues are highlyconserved and are important for receptor binding. Thus, additionalfragments or modified sequences can be easily made that exclude or alteramino acids of GLP-1 and exendin-4, other than these 5. Because thedifferentiation activity disclosed herein is easy to assess, thedetermination that a fragment is within the scope of the invention isroutine.

The present invention provides a population of insulin secreting cellsmade by the methods described herein. Because IDX-1 expression isrequired for insulin secretion from a cell, the non-insulin producingcells that can be used to make insulin secreting cells should includecells that express IDX-1 constitutively or upon stimulation with agrowth factor or by transfecting the cell with a nucleic acid encodingIDX-1 prior to, during, or after treatment of the non-insulin producingcells with the growth factor.

The present invention also provides a growth factor that candifferentiate insulin producing cells from non-insulin producing cells.Such growth factors include but are not limited to GLP-1, Exendin-4, orgrowth factors having amino acid sequences substantially homologousthereto, and fragments thereof. Differentiation could occur in vivo orin vitro upon contact of the non-insulin producing cell with the growthfactor. The contact could be one time by bolus, one time by continuousinfusion, or repeatedly by bolus or continuous infusion.

The present invention also provides a method of screening for growthfactors that differentiate an insulin producing cell from a non-insulinproducing cell. More specifically, the screening method involves thesteps of (1) contacting the growth factor to be screened with anon-insulin producing cell, (2) evaluating the non-insulin producingcell for characteristics of insulin producing cells, and (3) identifyingthe growth factors that differentiate insulin producing cells fromnon-insulin producing cells. The preferred characteristics of insulinproducing cells include the ability to transcribe the insulin gene, theability to translate the insulin mRNA, the ability to release or secreteinsulin, the ability to store insulin, the ability to sense levels ofglucose, and the ability to release insulin in a regulated fashion.Since expression of the transcription factors IDX-1, Beta 2/NeuroD, andE47 are believed to be necessary for production of insulin, thesefactors will also typically be expressed in the insulin-producing cellof the invention.

By “contacting” is meant an instance of exposure of the extracellularsurface of a cell to a substance at physiologically effective levels. Acell can be contacted by a growth factor, for example, by adding thegrowth factor to the culture medium (by continuous infusion, by bolusdelivery, or by changing the medium to a medium that contains growthfactor) or by adding the growth factor to the intracellular fluid invivo (by local delivery, systemic delivery, intravenous injection, bolusdelivery, or continuous infusion). The duration of “contact” with a cellor group of cells is determined by the time the substance, in this casea growth factor, is present at physiologically effective levels in themedium or extracellular fluid bathing the cell. GLP-1 has a shorthalf-life of several minutes, whereas Exendin-4's half-life issubstantially longer, on the order of hours. A bolus of GLP-1 would,therefore, have contact with the cell for minutes, and a bolus ofExendin-4 would contact the cell for hours.

The contacting step in the methods of the present invention can takeplace in vitro. For example, in a transplantation protocol, ex vivomethods can be employed such that non-insulin producing cells areremoved from a donor (e.g., the subject being treated) and maintainedoutside the body according to standard protocols well known in the art(see Gromada et al., 1998). While maintained outside the body, the cellscould be contacted with the growth factor and the cells subsequentlyinfused (e.g., in an acceptable carrier) or transplanted using methodswell known in the art into the donor subject or a subject different fromthe donor subject.

Alternatively, the contacting step of the present invention can takeplace in vivo. Methods for administering GLP-1, Exendin-4 or relatedgrowth factors are provided herein. The GLP-1, Exendin-4, or relatedgrowth factors are administered systemically, including, for example, bya pump, by an intravenous line, or by bolus injection (Gutniak et al.,1992; European Patent Application, Publication No. 0619322 A2; U.S. Pat.No. 5,614,492; U.S. Pat. No. 5,545,618). Bolus injection can includesubcutaneous, intramuscular, or intraperitoneal routes.

Non-insulin producing cells begin to differentiate into insulinproducing cells after about twenty-four hours of contact with GLP-1 orExendin-4, growth factors having amino acid sequences substantiallyhomologous thereto, or fragments thereof. The maximum number of cellsthat differentiate into insulin-producing cells usually have done soafter about seven days of contact. Interestingly, the new insulinproducing cells continue to show the capacity to produce insulin evenafter contact with GLP-1 or Exendin-4, their fragments or related growthfactors is discontinued. The new insulin producing cells show thecapacity to produce insulin at least up to 2 weeks after contact isdiscontinued.

Thus, the contacting step will typically be for at least twenty-fourhours. By “at least twenty-four hours,” is meant twenty-four hours orgreater. Specifically, the non-insulin producing cells can be contactedwith the growth factor for 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60 hours up to 3, 4, 5, 6, 7, or more daysor any particular intervening time in hours or minutes within the aboverange. Preferably the non-insulin producing cells will be contacted withthe growth factor for seven days.

The dosages of GLP-1, Exendin-4, their active fragments or relatedgrowth factors to be used in the in vivo or in vitro methods andprocesses of the invention preferably range from about 1pmoles/kg/minute to about 100 nmoles/kg/minute for continuousadministration and from about 1 nmoles/kg to about 40 mmoles/kg forbolus injection. Preferably, the dosage of GLP-1 in in vitro methodswill be 10 pmoles/kg/min to about 100 nmoles/kg/min, and in in vivomethods from about 0.003 nmoles/kg/min to about 48 nmoles/kg/min. Morepreferably, the dosage of GLP-1 in in vitro methods ranges from about100 picomoles/kg/minute to about 10 nanomoles/kg/minute, and in in vivomethods from about 0.03 nanomoles/kg/minute to about 4.8nanomoles/kg/minute. The preferred dosage of exendin-4 in in vitromethods is 1 pmoles/kg/min to about 10 nmoles/kg/mine, and in in vivofrom about 1 pmole/kg to about 400 pmoles/kg for a bolus injection. Themore preferred dosage of exendin-4 in in vitro methods ranges from about10 pmole/kg/minute to about 1 nmole/kg/minute, and in in vivo from about10 pmoles/kg to about 40 pmoles/kg for a bolus injection.

A method of differentiating non-insulin producing cells into insulinproducing cells, comprising contacting the non-insulin producing cellswith a growth factor selected from the group consisting of GLP-1 orExendin-4, growth factors having amino acid sequences substantiallyhomologous thereto, and fragments thereof is provided. By“differentiating non-insulin producing cells into insulin producingcells” is meant a change in the phenotypic characteristics of thenon-insulin producing cells such that the affected cells have at leastthe phenotypic characteristic of producing insulin. The affected cellmay have all of the phenotypic characteristics of a beta cell or mayhave less than all of the phenotypic characteristics of a beta cell. Theaffected cell may produce insulin but otherwise maintain the phenotypiccharacteristics of the non-insulin producing cell. For example, anon-insulin producing cell, such as a pancreatic amylase producing cell(i.e., pancreatic acinar cell), that is contacted with GLP-1 orExendin-4 can continue to express amylase, typical of an amylaseproducing cell, but, unlike the typical amylase producing cell, alsoproduces insulin. Thus, a continuum between complete phenotypic changeand a single phenotypic change is possible. The examples show thesurprising result that insulin producing capability can be conferredupon mature non-insulin secreting cells (e.g., acinar cells). Anincrease in proliferation of non-insulin producing cells may precede thedifferentiation of non-insulin producing cells into insulin producingcells, and “differentiating” is not meant to exclude any proliferationthat accompanies the change of the cell to an insulin producingphenotype.

Because of the importance of IDX-1, Beta 2/NeuroD, and E27 in thesecretion of insulin, the present invention also provides a method ofdifferentiating non-insulin producing cells into insulin producing cellsthat includes the additional step of transfecting the non-insulinproducing cell with a nucleic acid or nucleic acids encoding IDX-1, Beta2/NeuroD, and/or E27 prior to contacting the non-insulin producing cellwith GLP-1, Exendin-4, or similar growth factor. Alternatively, anadditional step can comprise transfecting a cell already contacted withGLP-1 or Exendin-4, or a similar growth factor with a nucleic acid ornucleic acids encoding IDX-1, Beta 2/NeuroD, and/or E27. If thecontacted cell is in vivo, transfection could be achieved by retrogradeperfusion of plasmid DNA for IDX-1 into the secretory duct of thepancreas (see Goldfine et al., 1997). Additionally, in some cases,expression or IDX-1, Beta 2/NeuroD, and E47 can result from theapplication of certain proteins to non-IDX expressing cells, including,for example, stem cells.

The present invention provides a method of enriching a population ofcells for insulin-producing cells, comprising contacting the populationof cells with a growth factor that promotes differentiation ofnon-insulin producing cells into insulin-producing cells. The populationof cells produced by this process is expanded in the number of insulinproducing cells and can be used in the treatment methods describedherein.

The present invention further provides a method of promoting pancreaticamylase producing cells to produce both insulin and amylase, comprisingcontacting the pancreatic amylase producing cells with a growth factorselected from the group consisting of GLP-1 or Exendin-4, growth factorshaving amino acid sequences substantially homologous thereto, andfragments thereof. An example of this method is provided in theexamples.

The present invention further provides a method of treating diabetes ina subject diagnosed with Type 1 diabetes, comprising administering tothe subject a growth factor selected from the group consisting of GLP-1,growth factors having amino acid sequences substantially homologousthereto, and fragments thereof by continuous infusion for at leasttwenty-four hours. Alternatively, the growth factor can be selected fromthe group consisting of Exendin-4, growth factors having amino acidsequences substantially homologous thereto, and fragments thereof. SinceExendin-4 has a fairly long half-life, compared to GLP-1, it can beadministered by bolus at least once. The treatment methods are effectiveto treat diabetes in a subject with Type 1 diabetes, because the growthfactor promotes the differentiation of non-insulin producing cells inthe subject into insulin producing cells, as described in detail herein.

The subject of the invention can include individual humans, domesticatedanimals, livestock (e.g., cattle, horses, pigs, etc.), pets (like catsand dogs).

By “diabetes” is meant diabetes mellitus, a metabolic diseasecharacterized by a deficiency or absence of insulin secretion by thepancreas. As used throughout, “diabetes” includes Type 1, Type 2, Type3, and Type 4 diabetes mellitus unless otherwise specified herein.

The present invention also provides a method of treating diabetes in asubject, comprising obtaining non-insulin producing cells from thesubject being treated or from a donor; contacting the non-insulinproducing cells with a growth factor in vitro, thereby promotingdifferentiation of non-insulin producing cells into insulin-producingcells; and administering the insulin-producing cells that were promotedto differentiate from non-insulin producing cells to the diabeticsubject. In the method of treating diabetes, wherein the non-insulinproducing cells are from a donor, the donor can be a cadaver. As afurther embodiment of the present invention, the non-insulin producingcells can be allowed to proliferate in vitro prior to contact with thegrowth factor. Preferably, promoting differentiation of non-insulinproducing cells into insulin-producing cells will result in greater thanabout 20% differentiation of non-insulin producing cells intoinsulin-producing cells. Even more preferably, greater than about 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% ofthe treated cells will differentiate into insulin-producing cells.

Altering the surface antigens of the insulin producing cells obtained bythe differentiation of the non-insulin producing cells into the insulinproducing cells, can reduce the likelihood that the insulin producingcells will cause an immune response. The cells with altered surfaceantigens can then be administered to the diabetic subject. The cellsurface antigens can be altered prior to, during, or after thenon-insulin producing cells are differentiated into insulin-producingcells.

The present invention also provides a method of differentiatingendothelial cells into smooth muscle cells, comprising contacting theendothelial cells with a growth factor selected from the groupconsisting of GLP-1 or Exendin-4, growth factors having amino acidsequences substantially homologous thereto, and fragments thereof.

The present invention will now be illustrated with reference to thefollowing examples.

EXAMPLES OF THE INVENTION Example 1

As GLP-1 in cultured insulinoma cells is known to impact positively oninsulin secretion, insulin synthesis and insulin messenger RNA, GLP-1'seffects on aging Wistar rats were evaluated.

Materials. GLP-1 and exendin [9–39] (Ex), a peptide receptor antagonistof GLP-1, were purchased from Bachem (King of Prussia, Pa.). Chemicalreagents were from Sigma (St Louis, Mo.), unless otherwise stated.

Animals. Three month (young) and 22 month (old) old Wistar rats from theWistar colony in the NIA (Baltimore, Md.) were used. They had beenmaintained on rat chow and fed ad libitum. All our aged rats are theoffspring of ten founder families maintained at the NIA.

Protocols. To insure that old animals were capable of responding toGLP-1, we carried out an acute experiment with an intravenous bolus ofGLP-1. Six old (22 months) and 6 young (3 months) Wistar rats werefasted overnight. Following anesthesia with 50 mg/kg pentobarbital, acatheter was placed in the femoral artery for blood sampling, and abolus of GLP-1 (0.2 nmol/kg) was given into the saphenous vein over 30sec. Blood (taken at 2, 4, 7, and 10 min) was collected for insulindetermination.

The rats were implanted with an Alzet micro-osmotic pump (Alza Corp.,Palo Alto, Calif.) in the interscapular region for 48 hrs. In thetreated group GLP-1 was delivered at the rate of 1.5 and Ex at 15pmol/kg⁻¹.min⁻¹. It has been shown that in order to prevent GLP-1'sinsulinotropic effect a 10-fold higher concentration of Ex is required(Wang et al., J. Clin. Invest., 1995). Control animals received normalsaline in their pumps and received their infusion for the same length oftime.

For glucose tolerance testing, after the pumps were in place 36 hrs (n=6each for control and GLP-1 treatment), the rats were fasted overnight,anesthetized with 50 mg/kg pentobarbital, and the pumps were removed,giving a total GLP-1 infusion time of 48 hrs. A catheter was placed inthe femoral artery for blood sampling and blood was collected for GLP-1measurement. An intraperitoneal (ip) glucose tolerance test (IPGTT,Ig/Kg BW) was administered 120 min after removal of the pumps. Bloodsamples were obtained at 15, 30, 45, 60, and 90 min in order to estimateglucose and insulin levels. Blood (200 μl) was drawn into heparinizedtubes containing EDTA for glucose and insulin determination.

For intraislet content of insulin, 14 rats were used. Seven were treatedwith GLP-1, and seven with saline, as described above. After 48 hrs,preceded by an overnight fast, the animals were sacrificed and islets ofLangerhans harvested as described previously (Perfetti et al., 1995;Egan et al., 1991). We then measured the intraislet insulin content in50 islets picked at random from each individual pancreas. Picked isletswere centrifuged, any residual medium was removed, the pellet wassuspended in ice-cold acid-ethanol (500 ul) and homogenized. Aftercentrifugation of the homogenate (1400×g, 4° C.) the supernatant wascollected for measurement of intracellular insulin. The pellet wasdissolved in formic acid and protein content was determined.

Assays. Plasma glucose was measured by the glucose oxidase method (Eganet al., 1991). Insulin and GLP-1 were measured by RIA as previouslypublished (Wang et al., Endocrinology, 1995; Nathan et al., 1992). Theamount of cellular proteins was measured using the Bradford method(Bio-Rad Richmond, Calif.) using bovine γ-globulin as standard.

RNA isolation and quantitation of endocrine pancreatic mRNAs.

Whole pancreata of rats that had been subjected to 48 hrs infusion withGLP-1 or saline were used to extract total RNA. After an overnight fast,animals were sacrificed, pancreata were removed and frozen in liquidnitrogen as quickly as possible. RNA was extracted by homogenization inguanidinium isothiocyanate, followed by ultracentrifugation on a 5.7 Mcesium chloride cushion (Glisin et al., 1974; Chigwin et al., 1979).Poly-A RNA was then prepared from total RNA by affinity chromatographyusing oligo (dT) columns (Biolabs INC, Beverly, Mass.). RNA wasquantified by spectrophotometric analysis at 260 nM. Slot-blot analysisusing poly-A RNA was used for quantitation of mRNA levels ofglucokinase, which is the main glucose sensor of the beta cell(Matschinsky, 1990), the three hexokinases, GLUT2, the major glucosetransporter of the beta cell (Mueckler, 1990), and insulin. Fourmicrograms of poly-A RNA were diluted in 50 μl TE buffer (Tris-HCL 10mM, EDTA 1 mM, pH 7.4), 20 μl of 37% formaldehyde and 20 μl of 10× SSC.Samples were incubated at 60° C. for 15 min and then diluted with 1 mlof ice-cold 10× SSC (1×SCC=0.15 M NaCl+0.015 sodium citrate). Each wellof slot-blot minifolder was rinsed once with ice-cold 10× SSC and 300 μlof sample per well was then loaded in triplicate onto the membrane.Vacuum was applied to drain the samples through the membrane, followedby three washes of the wells with ice-cold 10× SSC. Finally, membraneswere baked for 2 hrs at 80° C. in a vacuum oven for RNA crosslinking.

The hybridizations with cDNA probes (rat insulin II from Dr. S. J.Giddings, Washington University, St. Louis, Mo.; rat glucokinase fromDr. M. A. Magnuson, Vanderbilt University, Nashville, Tenn.; rat GLUT2from Dr. M. J. Birnbaum, Harvard Medical School, Boston, Mass.; andhexokinase I, II, III cDNAs from Dr. J. E. Wilson, Michigan StateUniversity, East Lancing, Mich.) were carried out as previouslydescribed (Wang et al., Endocrinology, 1995; Wang et al., Mol. Cell.Endocrinol., 1996). All cDNA probes were labeled with [³²P] dCTP(Amersham Life Science, Arlington Heights, Ill.) by random primingprocedure using Sequenase (United States Biochemical, Cleveland, Ohio).An oligonucleotide homologous to the poly-A tail of mRNAs wassynthesized on an Applied Biosystem DNA synthesizer(5′GATGGATCCTGCAGAAGCTTTTTTTTTTTTTTTTTTTT3′) and used to quantify totalcellular mRNA. Hybridization with oligo dT₂₀ was carried out in order toverify that an approximately equal amount of RNA was used for eachsample. Oligonucleotide probes were end-labeled with [³² P] γATP(Amersham) using T4 polynucleotide kinase (New England Biolabs, Beverly,Mass.). The hybridizations with oligonucleotide probes were carried outas described before (Wang et al., Endocrinology, 1995; Wang et al., Mol.Cell. Endocrinol., 1996) and quantified using a Betascope 603 blotanalyzer (Betagen, Walthman, Mass.).

RNA isolation and quantitation of mRNAs in islets of Langerhans. Toconfirm the changes seen in whole pancreata RNA was isolated from isletsof Langerhans from animals treated as described above with GLP-1. Isletswere isolated and RNA extracted using the micromethod, previouslydescribed (Perfetti et al., 1995). Approximately 5 μg of total islet RNAwas from one pancreas. Slot-Blot analysis was carried out to quantitatemRNA levels of the hexokinases, GLUT2, and insulin.

Statistical analysis The data were expressed as the mean±SEM.Significance of the insulin and glucose data obtained from the IPGTTwere tested using repeated measures analysis of variance by SAS (SASInstitute Inc.; Cary, N.C.). If a significant interaction was documented(p<0.05), values at single time points were compared by non-pairedStudent's t test. All other data were analyzed using the non-pairedStudent's t test: a p<0.05 was judged as significant.

Response to acute iv GLP-1 bolus. Old and young fasted animals respondedequally well to a bolus of 0.2 nmol/kg GLP-1 delivered over 30 sec.Their insulin responses were superimposable (FIG. 1). At 2 min aftercompletion of the bolus the insulin response was maximal in both young(373.3±43.7 pmol/1) and old (347.7±25.7 pmol/l) animals and in bothgroups insulin levels had returned to baseline at 10 min.

Glucose tolerance testing. Old animals have frank glucose intolerancewhen compared to young animals during an IPGTT (FIG. 2). Fastingglucose, taken just prior to the ip glucose, was not different betweentreated and control animals. Blood glucose was significantly lowerduring the glucose tolerance test in the animals treated with GLP-1 whencompared to control animals at the 15 (9.04±0.92 vs 11.61±0.23 mmol/l)and 30 (8.61±0.39 vs 10.36±0.43 mmol/l) min time points (FIG. 2). Theold animals were also no longer glucose intolerant when compared withyoung animals. On reviewing the insulin response at the same time it canbe seen that the 15 min insulin response was significantly better in theGLP-1-treated animals compared to the controls (FIG. 3). Indeed, thesaline-treated rats had their peak insulin level at 30 min, while theGLP-1-treated animals peaked at 15 min. This brisk insulin responseaccounted for the drop in blood glucose in the treated animals (FIG. 2).Overnight fasting levels of insulin were higher in the GLP-1-treatedanimals but due to great intranimal variation this was not statisticallydifferent from controls (192±47 vs. 134±45 pmol/l). 48 hr infusion ofGLP-1 in 22 month old Wistar rats potentiates insulin response to anIPGTT. This phenomenon is observed even after termination of the GLP-1infusion, indicating that GLP-1 is capable of inducing long-term changesthat go over and beyond modulating insulin release. The major change inthe insulin-response curve was in early insulin release after theglucose load, and was induced by a shift in the maximum insulinsecretion from 30 min after glucose injection, as observed in controls,to only 15 min in the GLP-1 treated rats.

Intraislet insulin content. There was variation in the amount of insulinbetween the islets from each individual pancreas as might be expectedfrom aged animals (FIG. 4). However, there was consistently more insulinin the islets from treated animals (p<0.01). Islets from controls andGLP-1 treated rats had 5.31±1.19 vs. 19.68±3.62 ng of insulin per ug oftotal pancreatic proteins, respectively.

GLP-1 plasma levels. We measured plasma GLP-1 in 3 animals 6 hrs aftercommencement of the GLP-1 infusion to insure both that steady stateGLP-1 levels were reached and to verify that the peptide was actuallybeing infused. Plasma GLP-1 level at 6 hrs was 106.7±17.6 while at 48hrs it was 125.0±41.4 pmol/l. Before the commencement of the glucosetolerance testing plasma GLP-1 was below the level of detectability ofthe assay. Fasting GLP-1 levels in control Wistar rats were 10–20pmol/l. There was no difference in the fasting levels of GLP-1 betweenthe young and old animals. Therefore, our infusion of GLP-1 raisedplasma GLP-1 levels to approximately 6 times the fasting levels. Sincefed levels in Wistar rats (Wang et al., J. Clin. Invest., 1995), as wellas humans (Gutniak et al., 1992), are reported to approximately doubleafter eating, the plasma levels attained with the pumps werepharmacological.

Effect of GLP-1 on gene expression. We measured the abundance of insulinmRNA as well as mRNA levels of other factors involved in the early stepsof glucose-mediated insulin release as well as glucose metabolism inbeta cells. Results were quantified by densitometry, normalized by usingoligo dT hybridization and expressed in relative terms by assigning theyoung control result a value of 1. FIG. 6 shows the blots for insulinmRNA from whole pancreata in 6 young and 12 old animals ans combinedresults from all the animals shown in FIG. 5. FIG. 7 shows the blots ofthree isolated islet RNA preparations from old animals.

The levels of insulin mRNA were increased approximately 50% in old vsyoung animals (FIG. 5A, P<0.05, and FIG. 6). GLP-1 increased the insulinmRNA in both young and old animals compared to controls (FIG. 5A, P<0.01and FIG. 6). Similar results can be seen in the isolated isletpreparations (FIG. 7). This increase was entirely prevented when theanimals were treated simultaneously with Ex, an inhibitor of GLP-1binding to its own receptor. Of great interest is the fact that in theanimals treated with Ex alone or Ex with GLP-1, the insulin mRNA levelswere lower than in controls (p<0.05). Insulin mRNA levels fell anaverage of 60% in the presence of Ex alone.

GLUT-2 mRNA levels in old animals were decreased by 70% compared withyoung controls and this was entirely reversed by GLP-1 treatment (FIG.5B, P<0.001). The increase in GLUT2 mRNA levels in old animals by GLP-1can be seen in both islet (FIG. 7) and whole pancreatic preparations(FIG. 6). in the young animals, GLP-1 did not significantly influencethe GLUT2 mRNA levels (FIG. 5). The levels fell by 50% in the presenceof Ex alone (FIG. 5B, P<0.05), but not in the animals treated with Exand GLP-1 (FIG. 5B).

There were no differences between young and old animals in glucokinasemRNA levels (FIG. 5C). GLP-1 significantly increased glucokinase levelsin young (FIG. 5, P<0.05), but much more so in old animals (FIG. 5C,P<0.001, FIG. 7). Similar results were seen in the old animals with theisolated islet preparations (FIG. 7). Ex completely preventedGLP-1—induced increases in glucokinase mRNA.

For all preparations, the results from the pancreata were reflected inthe islets. Hexokinase I, II and III mRNA levels were very low in thewhole pancreata and islets and did not appear to be altered by GLP-1treatment. We also infused GLP-1 for 5d into old rats (n=6) and foundthe same results as with the 48-h infusion.

Following continuous infusion with GLP-1, the pancreata weresurprisingly larger than control pancreata. The pancreata of treatedanimals weighed 26% more than the pancreata of control animals.

Also, surprisingly, insulin secretion remained improved even afterremoval of the exogenous source of GLP-1. The biological half-life ofGLP-1's insulinotropic action in blood is 6–8 min (Elahi et al., 1994)and since GLP-1 infusion had been terminated at least 2 hours prior toperforming the glucose tolerance testing the continued presence ofelevated GLP-1 levels, at least in the short-term, was not necessary forthe improvement in glucose tolerance in the aging Wistar animals.

GLP-1 increases insulin biosynthesis and insulin mRNA levels in vivo, aspreviously shown in insulinoma cells (Wang et al., Endocrinology, 1995).GLP-1 would also appear to be necessary for the normal maintenance ofmRNA levels of insulin in the pancreas. Not only did Ex inhibit theGLP-1 effect on insulin mRNA, but it also caused a decrease in insulinmRNA in animals given Ex alone. Ex is a competitive inhibitor of GLP-1binding to its receptor, a 10-fold higher concentration of Ex beingrequired to inhibit GLP-1's insulinotropic effect (Wang et al., J. Clin.Invest., 1995), so presumably it was inhibiting the binding ofendogenous GLP-1 in the animals that received Ex alone. This means thatGLP-1 has effects on maintaining insulin mRNA levels in thephysiological range.

It has been proposed that in Type 2 diabetes the beta cell stores ofinsulin fall below a critical level, and that this causes a subsequentreduction in glucose-induced insulin responses (Hosokawa et al., 1996).As our data show, GLP-1 is capable of increasing intraislet insulincontent and when given continuously, rather than just by bolus, may alsoinduce changes beneficial to beta cell function, over and beyond itseffects on simply insulin secretion.

Example 2

Exendin-4 is a peptide produced in the salivary glands of the GilaMonster lizard. In the present example, we report that in Wistar rats,bred in the National Institute of Aging (NIA), it was a far more potentinsulinotropic agent in several ways than is GLP-1. We further reportthat exendin-4 leads to sustained improvement of diabetic control in arodent model of type 2 diabetes.

Materials. Exendin-4 and GLP-1 were purchased from Bachem (King ofPrussia, Pa.). Chemical reagents were from Sigma (St Louis, Mo.), unlessotherwise stated.

Animals. Four month old Wistar rats from the Wistar colony in the NIA(Baltimore, Md.) were used for the acute experiments of the effects ofexendin-4 and GLP-1 (see Example 1). They had been maintained onstandard lab chow and fed ad libitum. For the long-term experiments,diabetic mice (C57BLKS/J-Lepr^(db)/Lepr^(db)) lacking the leptinreceptor, and their non-diabetic littermates were purchased at 4 weeksof age from Jackson Laboratories (Bar Harbor, Me.). They were housed twoper cage and also were fed ad libitum. The same mice were caged togetherfor the duration of the study. Wistar rats are caged on wire while thebedding for the mice was a paper based product, “Carefresh” (AbsorptionCo., Bellingham, Wash.).

Protocols. Wistar rats were fasted overnight. Following anesthesia with50 mg/kg pentobarbital, a catheter was placed in the femoral artery forblood sampling. A bolus of either exendin-4 or GLP-1 was given into thesaphenous vein (iv) over 30 sec to 12 animals, while a bolus of normalsaline (NaCl) was given to the other six. The order of the injectionswas rotated. Blood (taken at −5, 0, 2, 5, 15, 30, 60, 120, and 180 min)was drawn into heparinized tubes containing EDTA and aprotinin forinsulin determination (See Example 1). Animals were acclimated to thefacility for at least 2 days.

Eleven diabetic and 10 non-diabetic animals received 24 mmol/kgexendin-4 ip daily thereafter (7–9 am) while 10 diabetic and 10non-diabetic animals received ip NaCl. Subsequently, whole blood glucoselevels, taken from a retro-orbital sinus, were determined in the miceusing a Glucometer Elite (Bayer). This regimen was continued for 12–13weeks. On day eight two of the non-diabetic mice (cage-mates) and dayfourteen one of the diabetic mice died just after receiving exendin-4.Animals were weighed weekly. After one week of the regimen blood sampleswere again taken from a retro-orbital sinus for determination of insulinand glucose levels. At the end of the regimen, fasting blood sampleswere obtained for glucose and insulin levels and whole blood containingEDTA was assayed for hemoglobin A1c (Hb A1c) on the same day from thefour groups.

In another group of eight diabetic mice 14 weeks old we gave 24 nmol/kgexendin-4 ip daily and NaCl ip daily for five days to four each of thegroup.

cAMP determinations. Islets of Langerhans were harvested from Wistarrats (Perfetti et al., 1995) and batches of 25 islets were thenincubated for 1 h at 37° C. in a buffer containing (mM) 140 NaCl, 5 KCl,1 NaPO₄ 1 MgSO₄, 5 glucose, 2 CaCl₂, 20 HEPES, buffer (pH 7.4) and 0.1%bovine serum albumin. Following this, they were incubated in the samebuffer for 1 h in the presence of GLP-1 (1 nM) or exendin-4 (1 nM). Somebatches of islets were then washed three times with ice-cold phosphatebuffer saline (PBS) and lysed with 1 ml ice-cold 0.6 mM perchloric acid.Other batches were washed three times in the buffer at 37° C. to removepeptide and left another 15 min before being washed three times inice-cold PBS followed by lysis with the perchloric acid. The lysates(950 μl) were then transferred to microcentrifuge tubes and cAMPmeasured as previously described (See Example 1) using a cAMP [³H] assaykit (Amersham). Cellular protein was assayed using the Bradford method(Bio-Rad, Richmond, Calif.) using bovine γ-globulin as standard.

Assays. Plasma glucose was measured by the glucose oxidase method (Wanget al., 1997). Insulin was measured by RIA as previously described (SeeExample 1). Hb A1c was assayed using a BIO-RAD (Herculas Calif.) DiaSTATmachine which uses low pressure cation exchange chromatography inconjunction with gradient elution to separate hemoglobin subtypes andvariants from hemolyzed blood. The separated hemoglobin fractions weremonitored by means of absorption of light at 415 nm.

Statistical methods. All results are given as mean±SEM. T tests werebased on the results of an F test that looked at the equality ofvariance of the two means. If the variances were statisticallysignificantly different then the t test was based on unequal variances.For determination of the EC₅₀ the basal plasma insulin levels weresubtracted and the remaining activity at each concentration wasexpressed as a percent of the maximal activity (achieved by an excess ofpeptide). This was then transformed into a logit format where logit=ln(% activity/[100-% activity]) and was plotted as a function of the logconcentration of the compound.

Exendin-4 effects in Wistar rats. Exendin-4 was more potent aninsulinotropic agent than GLP-1 on several levels when givenintravenously. Maximal insulin response in our Wistar rats is seen with0.4 nmol/kg GLP-1 (De Ore et al., 1997). At this same exendin-4concentration maximal insulin response is approximately doubled (FIG.8). Insulin levels return to baseline by 10 min with GLP-1, but withexendin-4 they actually go below baseline and have returned to baselineby 60 min. EC₅₀ concentration for insulin release is lower and themaximum amount of insulin secreted by exendin-4 is higher than withGLP-1. EC₅₀ was 0.019 (FIG. 9) versus 0.19 mmol/kg for exendin-4 versusGLP-1 (See Example 1), respectively. The animals given exendin-4 had anobvious increase in urine output for the duration of the study (we didnot quantitate the volume of urine) as they continued to urinatefrequently during the study despite the blood drawing which would bediminishing circulating blood volume, while the GLP-1-treated animalsurinated little if any during the study.

Effects of exendin-4 and GLP-1 on cAMP levels in isolated islets.Exendin-4 increased cAMP levels more in isolated islets than GLP-1 atequimolar concentrations. GLP-1 increased cAMP in aconcentration-dependent manner with maximum cAMP response at 1 nM. Atthat concentration of exendin-4 cAMP levels were approximately 3-foldhigher (FIG. 10) than with GLP-1. This probably explains why exendin-4causes a higher maximal insulin release than GLP-1. In an effort to seeif exendin-4 or GLP-1 might remain on the GLP-1 receptor and so maintainan increase in cAMP after the removal of the peptides from the buffersolution we removed the peptide from some islets by three washes infresh buffer and then measured cAMP after 15 min. With both peptides,cAMP levels returned to baseline at least by 15 min.

Effects of chronic treatment with exendin-4 in mice. The biologicalactivity of exendin-4, as measured by its ability to lower blood sugar,was much longer than we expected when given ip or subcutaneously todiabetic animals. In preliminary experiments we found thatexendin-4-treated diabetic mice had lower blood sugars 24 hours after ipand subcutaneous (sc) injections while with GLP-1 injections, bloodsugars were back to baseline. This lead us to design a long-termexperiment with exendin-4. At the initiation of the daily ip exendin-4regimen in the mice fasting blood glucose was 145±51 mg/dl in thenon-diabetic mice and 232±38 mg/dl in the diabetic mice. After one weekof treatment the fasting glucose level in the exendin-4-treatednon-diabetic mice was 70±25 mg/dl and was significantly lower than inthe NaCl-treated non-diabetic animals, 135±5 mg/dl (p<0.05). Thediabetic animals had a highly significant response to exendin-4. Glucoselevels dropped to 90±11 mg/dl in the exendin-4-treated animals from238±51 mg/dl (p<0.002) in the NaCl-treated animals (Table 1). Wemeasured fasting insulin levels in the diabetic animals that receivedNaCl or exendin-4. They were higher in the animals that receivedexendin-4 (p<0.002). On the basis of these data, we continued to treatthe animals daily with exendin-4. As the bedding was a paper-basedproduct which turns progressively darker with increased urination, itwas clear that the cages of the exendin-4-treated diabetic animals,while not totally dry as were the non-diabetic cages, always wereobviously drier 24 hours after changing than the cages of theNaCl-treated diabetic animals (FIG. 11, photograph taken after 9 weekstreatment). We surmised that the decreased urination in theexendin-4-treated diabetic mice was due to less osmotic diuresis becauseof lower blood glucose.

The diabetic animals were clearly heavier than the non-diabetic animals.After 9 weeks the weight of the non-diabetic animals reached a plateauat approximately 28 grams (g) while the diabetic animals continued togain weight. At 13–14 weeks of treatment the NaCl-treated animals beganto lose weight (38.7 g) while the exendin-4-treated animals maintainedtheir weight (46.7 g).

We assayed the whole blood of the saline and exendin-4-treated animalsfor Hb A1c determinations and we measured plasma for glucose and insulinconcentrations after an overnight fast (FIG. 12). It can be seen thatall these parameters were significantly altered by the daily exendin-4treatment. Hb A1c was 8.8% in the NaCl-treated diabetic animals vs 4.7%in the exendin-4-treated animals (p<0.0001). Hb A1c was also lower inthe non-diabetic animals, 3.5 vs. 3.1% (p=0.0002), exendin-4-treated vs.NaCl-treated, respectively. Glucose levels were significantly lower(278.7±30.0 vs. 517±59 mg/dl, p<0.005) and insulin levels significantlyhigher (4,600±1,114 vs. 707.2±169.7 pmol/l, p<0.02) in theexendin-4-treated diabetic animals (FIG. 13). The trends in glucose andinsulin were the same in the non-diabetic animals with exendin-4treatment though not as dramatic (FIG. 13).

In the eight diabetic mice that were 14 weeks old when their treatmentbegan, blood sugars were 640±37 mg/dl in the NaCl-treated animals and355±21 mg/dl in the exendin-4-treated animals after five days. Theirinsulin levels were 6,904±705 vs. 1,072±54 pmol/l, exendin-4-treated vs.NaCl-treated, respectively.

Following sc and ip exendin-4, blood sugars in diabetic mice had a muchmore prolonged biological response in that the blood sugars stayed lowerlonger (up to 24 hours after an ip dose) than was expected from theinsulin response to the iv exendin-4, with the glucose lowering effectof the ip injections being less variable than sc. This was possibly dueto greater variation in the sc technique and perhaps even loss of thepeptide during injections in some cases.

In the studies involving ip injection of exendin-4 ip for one weekdaily, the fasting blood sugars as a result of just one injection a daywere actually lower than the blood sugars of the non-diabetic animals.It was also obvious looking at the cages each morning which diabeticanimals were receiving exendin-4 as their cages were always drier. Thiseffect was also seen in the non-diabetic animals that receivedexendin-4.

In the experiments where exendin-4 was given daily for five days todiabetic animals whose mean blood sugars were 640 mg/dl, there was alsoa marked effect on lowering blood sugar, and insulin levels weremarkedly increased. This is unlikely due to expansion of the beta cellmass and would suggest that beta cells of diabetic rats are stillresponsive to exendin-4 even in the face of such marked hyperglycemia.

Even after 12 weeks of exendin-4, blood sugars were lower than in theNaCl-treated animals. Typically primary antibody responses postintraperitoneal injections take up to 14–21 days and exendin-4 is a weakhapten at any rate. So for the first several weeks of treatmentexendin-4 would not be expected to be neutralized by antibody. Inconjunction with this, exendin-4's biological effects are at very lowconcentrations and therefore possibly the peptide has a higher affinityfor the GLP-1 receptor than for its specific antibody and so might notbe totally neutralized by antibody. Other possibilities as to whyexendin-4 might not be neutralized are that there is an exendin-4-likepeptide produced in rodents that are not yet identified or that anexendin-4-type molecule is made in utero in rodents which would rendermature animals exendin-4-tolerant.

We have given exendin-4 at a lower concentration (1 nmol/kg) ip for oneweek to diabetic mice. It was just as effective at lowering blood sugaras at the larger amount reported in this example.

We saw no untoward effects of the daily injections on the behavior ofthe mice. We have since observed that for the first 3–4 days ofinjection of exendin-4 the weight of the animal drops but by the seventhday it is back to the same as NaCl-treated animals. In our long-termstudy we weighed weekly and so missed the initial drop. Except for thebedding being obviously drier each morning in the treated diabeticanimals, we could not detect any glaring deleterious effects ofexendin-4 on the animals. We therefore suggest that exendin-4 might besuperior to GLP-1 as a treatment for Type 2 diabetes in humans.

Example 3

Using the protocol of Example 1, GLP-1 was administered by continuousinfusion for one to five days to young and old rats, whereas controlrats received comparable saline infusions. Exendin-4, in contrast, wasadministered intraperitoneally one time daily for five days according tothe protocol of Example 2.

Approximately 20% of the cells in the GLP-1 treated pancreata werepositive for PCNA at five days. At the same time point, there wereproliferating cells in the islet. In addition, there were proliferatingcells lining the ducts and, more surprisingly, in the acinar tissue, anarea generally considered to be devoid of stem cells. Also surprisingly,a number of insulin positive cells were found outside the islets amongthe acinar tissue, where insulin positive cells are not expected.

These results show that continuous infusion with GLP-1 or repeatedintraperitoneal injection with Exendin-4 for at least two days resultsin an increase in total number of insulin positive cells and indifferentiation of acinar cells into insulin, IDX-1 positive cells.These results further suggest that GLP-1 and Exendin-4 increase theproliferation of cells and, specifically, an increase in proliferationof insulin producing cells within the acinar tissue.

Continuous infusion of GLP-1 promotes differentiation of non-insulinproducing cells into insulin producing cells upon contacting thenon-insulin producing cells with GLP-1 for greater than twenty-four. Theeffect was observed as early as 1 day, and the maximal effect as earlyas seven days. Such differentiation was surprising since the prior artshowed only insulinotropic results in beta cells. Furthermore, thepresent invention was surprising because acinar cells, which have neverbeen shown to be capable of producing insulin, are promoted to secreteinsulin upon contact with GLP-1. The increased number of insulinproducing cells remains unchanged for at least two weeks after treatmentis discontinued. Since differentiation to an insulin producing cell is aterminal event, de-differentiation back into a non-insulin producingcell at even later time points is unlikely. Thus, the effect ispermanent.

Exendin-4 is shown in the present invention to have the same effects asGLP-1 on the differentiation of insulin producing cells from non-insulinproducing cells. Surprisingly, Exendin-4 is shown to have a much longerhalf-life than GLP-1. The increase in the number of insulin producingcells, thus, can be achieved with daily bolus injection, rather thancontinuous infusion, of Exendin-4 for two days. After two injections,insulin producing cells outside the islet are observed. The maximaleffect is achieved by seven days. As with GLP-1, the effect persists forat least two weeks, and probably permanently, even after contact withthe Exendin-4 is discontinued.

GLP-1, Exendin-4, growth factors having amino acid sequencessubstantially homologous to GLP-1 or Exendin-4, and fragments thereofaffect the differentiation of non-insulin producing cells in vivo and invitro. Furthermore, a variety of non-insulin producing cells, includingstem cells and acinar cells can be promoted to differentiate intoinsulin producing cells. These advances over the prior art providemethods of treating diabetes mellitus whereby insulin producing cellsare increased in number by administration of the growth factor to apatient or by contacting the non-insulin producing cells in vitro.

Example 4

The purpose of this study was to determine if GLP-1 and the islethormones, glucagon and insulin, have effects on acinar tissue. We usedthe AR42J cells (Christophe, 1994), which are derived from a ratpancreatic exocrine tumor, as a model of acinar tissue. We then lookedat some aspects of the signal transduction system through which GLP-1 isalready known to work in beta cells (Goke et al., 1993; Holz et al.,1995; Yada et al., 1993).

Materials. GLP-1, glucagon, exendin-4 and exendin 9–39 were obtainedfrom Bachem (Torrance Calif.). Cholecytokinin (CCK), insulin, genesteinand vanadate were from Sigma Chemical Co (St. Louis, Mo.). The ratpancreatic cell line, AR42J, was from American Type Culture Collection(Rockville, Md.). Anti-tyrosine antibodies were purchased from UpstateBiotechnology, Inc (Lake Placid, N.Y.).

Cell Culture. AR42J cells were maintained in Dulbecco's modified Eagle'smedium (Gibco, Grand Island, N.Y.) (DMEM) supplemented with 10% FetalCalf Serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mMglutamine. Cells from passage 23–36 were used throughout this study.Cells were routinely plated at about 10⁵ cells/ml in 12-well clusterdishes and incubated in a humidified incubator at 37° C. with 95% airand 5% CO₂. As AR42J cells responded poorly to CCK we routinelyincubated the cells with 10 nM dexamethasone for 48 h before use as thiswas known to induce CCK responsivity in a concentration-dependent manner(Logsdon et al., 1987).

Amylase Assay. For amylase secretion, cells were washed free of mediumwith 2 ml phosphate buffered saline (PBS). Incubation was then carriedout in DMEM containing 15 mM HEPES, 0.2% bovine serum albumin (BSA) and0.01% soybean trypsin inhibitor. The hormones and reagents of interestwere added for 50 min at 37° C. The incubation medium was thenimmediately removed for amylase determination and the cells were againwashed in 2 ml ice-cold PBS. Lysate buffer containing (in mM) 130Tris-HCl, 10 CaCl₂, 75 NaCl, and 0.2% Triton X-100 (pH 8.0) was added tothe cells and the lysates were then collected for total amylase activity(Ceska et al., 1969). The released amylase was expressed as thepercentage of the total amylase activity of the cells.

Measurement of cAMP. Cells were grown in 12-well dishes and treated withhormones and reagents±IBMX as described above. They were then washed 3times in ice-cold PBS and lysed with 1 ml ice-cold 0.6 mM perchloricacid. The lysates (950 μl) were transferred to microcentrifuge tubes andthe pH adjusted to 7.0 using 5 M K₂CO₃. After centrifugation for 5 minat 10⁴ rpm, the supernatant was vacuum dried, and then recovered in 200μl Tris/EDTA buffer. After addition of 0.15 mM Na₂CO₃ (50 μl) and 0.15mM ZnSO⁴ (50 μl), followed by incubation for 15 min on ice, the saltprecipitate was removed by centrifugation for 15 min at 3.5×10³ rpm and50 μl of supernatant was assayed using a cAMP [³] assay kit (AmershamCorp., Arlington Heights, Ill.), (Steiner et al., 1972). Cellularprotein was measured using the Bradford method (Bio-Rad, Richmond,Calif.) with bovine gamma-globulin as standard (Bradford, 1976).

Measurement of intracellular calcium, [Ca²⁺]_(l). AR42J cells wereloaded with the fluorescent Ca²⁺ probe, indo-1 acetoxymethyl ester(indo-1/AM). The loading solution consisted of 50 μg of indo-1/AM(Molecular Probes Inc.), 30 μl of dimethyl sulphoxide (DMSO) and 5 μl of25% (w/w in DMSO) Pluronic F-127 (BASF Wyandott Corp.) This mixture wasadded to 2.0 ml of cells in Hank's balance salt solution (final indo-1concentration of 25 μM) and gently mixed on a shaking plate for 1 h. Thecells were then centrifuged at 400×g for 60 sec, resuspended in standardbathing solution, consisting of (in mM): 137 NaCl; 5 KCl; 1.3 MgSO₄; 5CaCl₂; 20 HEPES; pH adjusted with NaOH to 7.4, and stored for at least 1h before use. Both loading with indo-1 and the experiments were carriedout at room temperature (22–24° C.). The cell suspension was placed in achamber on the stage of an inverted fluorescence microscope (Spurgeon etal., 1990). The emission field was restricted to a single cell. Indo-1was excited at 350±5 nm every 5 ms, and the fluorescence emission wassplit into wavelength bands of 410±5 and 490±5 nm. The 410:490fluorescence ratio (ratio F410/F490), corrected for auto fluorescence,was used as an index of [Ca²⁺]_(l), using the methodology well know inthe art (Spurgeon et al., 1990). The cell autofluorescence was assessedin a large number of indo-1 non-loaded cells from the same batch. In atypical experiment, the standard bathing solution was exchanged rapidly(<200 ms) with one of the test solutions injected from a micropipetteplaced in close vicinity to the cell (Janczewski et al., 1993; Konnerthet al., 1986). Routinely, the cells were exposed to the test solutionfor 240–300s. Thereafter, the test solution was washed out, while[Ca²⁺]_(l) was monitored for additional 120–180s. The test solutionswere prepared just prior to the experiment by adding the hormones instandard bathing solution.

GLP-1 binding. AR42J cells were plated and cultured as described above.At the start of the binding experiment the cells were incubated withserum-free DMEM for 2 h at 37° C. Cells were then washed twice with 0.5ml binding buffer containing (in mM) 120 NaCl, 1.2 MgSO₄, 13 sodiumacetate, 5 KCl, 10 Tris, pH 7.6. Cells were then incubated overnight at4° C. with 0.5 ml binding buffer supplemented with 2% BSA, 500 U/mlaprotinin, 10 mM glucose, a range of concentrations of GLP-1 (0.03nM–100 nM) and 30,000 cpm ¹²⁵I-GLP-1 (2,000 Ci/mmol, Peninsula, Belmont,Calif.). We only used freshly prepared ¹²⁵I-GLP-1 within two weeks ofthe reference date. At the end of the incubation, the supernatant wasdiscarded and the cells were washed three times with ice-cold PBS. Cellswere lysed with 0.5 ml 0.5 N NaOH/0.1% SDS for 30 min at roomtemperature. The radioactivity was measured in the lysates in an ICNApec series gamma counter. Specific binding was determined bysubtracting the non-specific binding present at 500 nM GLP-1 from totalbinding. This method has been used previously to characterize GLP-1binding in CHO cells overexpressing GLP-1 receptor and in 3T3-L1adipocytes (Montrose-Rafizadeh et al., J. Biol. Chem., 1997;Montrose-Rafizadeh et al., J Cell Physiol., 1997).

RT-PCR of the GLP-1 Receptor. Complementary DNA was synthesized fromtotal cellular RNA using Maloney murine leukemia virus reversetranscriptase (Bethesda Research Laboratories, Gaithersburg, Md.) andrandom hexanucleotide primer (Pharmacia LKB Biotechnology Inc.,Piscataway, N.J.). PCR amplification (30 cycles) was performed (Saiki etal., 1997) from first strand cDNA using recombinant Taq DNA polymerase(Amplitaq, Perkin-Elmer, Cetus). Oligonucleotide primers were on 5′- and3′-end of the pancreatic GLP-1 receptor sequence (Thorens, 1992),^(5′)ACAGGTCTCTTCTGCAACC^(3′) and ^(5′)AAGATGACTTCATGCGTGCC^(3′),respectively. PCR products were then resolved on a 1% agarose gel andvisualized using ethidium bromide. The PCR products were subcloned inpBluescript vector and sequenced using the chain termination techniqueand Sequenase 2.0 kit (United States Biochemicals, Cleveland, Ohio). Thespecificity of the PCR product was also determined by the Bstx1restriction enzyme.

Immunoprecipitation and Western blotting of the GLP-1 receptor. AR42Jcells and an insulinoma cell line, RIN 1046-38 cells, were grown in 60mm dishes as described above. When the cells reached 80% confluence,they were washed twice with Krebs-Ringer buffer containing 115 mM NaCl,5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 24 mM NaHCO₃, and 25 mM HEPES andfrozen in liquid nitrogen. The frozen cells were scraped and lysed inRIPA buffer containing 20 mM Tris-HCl: pH 8.0, 137 mM NaCl, 1% TritonX-100, 0.5% deoxycholate, 0.1% SDS, 0.2 mM PMSF, 10 μg/ml leupeptin, 20μg/ml aprotinin, 1 mM Na-orthovanadate, 1 mM benzamidine. Insolublematerial was removed by centrifugation at 15,000×g for 15 min at 4° C.and the supernatant was collected for immunoprecipitation and WesternBlotting. Anti-GLP-1-R antibody against the N-terminal (gift from Dr.Joel Habener, Massachusetts General Hospital, Mass.) at 1:250 was addedto each tube, together with 40 μl protein A and protein G. Theimmunoprecipitation was carried out at 4° C. overnight and theimmunocomplexes were washed twice with RIPA buffer, rewashed another twotimes with washing buffer (25 mM Hepes, 0.1% Triton X-100 and 1 mMNa-orthovanadate), then the immunocomplex pellets were solubilized in 50μl of SDS-PAGE sample buffer at 70° C. for 10 min. Theimmunoprecipitated proteins were eluted with mini-resin column andsubjected to 4–20% SDS-polyacrylamide gel. After the gel waselectrotransferred to PVDF membrane, the blot was blocked with 5%non-fat milk in TBST buffer (20 mM Tris-HCl [pH 7.5], 137 mM NaCl and0.1% Tween 20) for 1 h at room temperature, and then incubated withantibody to GLP-1-receptor at 1:1500 for 1 h at room temperature. PVDFmembranes were washed three times with TBST and incubated withhorseradish peroxidase-conjugated anti-rabbit secondary antisera for 1 hat room temperature. After a series of washes in TBST, the blots weredeveloped using the EC1 chemiluminescent detection system.Autoradiographs were quantified using Image-Quant™ software (version3.3) on a Molecular Dynamics laser densitometer. In this experiment, theinsulin producing cell line RIN1046-38 cells was used as a positivecontrol for the presence of the GLP-1 receptor. Aliquots (20 μl) ofclarified cell lysates were used to determine protein concentrationwhich was estimated by the Bradford method (Bradford, 1976).

Tyrosine Phosphorylation Studies. AR42J cells were preincubated inKrebs-Ringer Balanced Buffer (KRBB) containing 115 mM NaCl, 5 mM KCl,2.5 mM CaCl₂, 1 mM MgCl₂, 24 mM NaHCO₃, and 25 mM HEPES for 2 h at 37°C. Then the medium was removed and fresh KRBB was added, followed byplacing the cell on a 37° C. hot plate for 5 min. After addition ofvarious reagents (see FIG. 24) for 5 min the reaction was terminated bysubmersion of the dishes in liquid nitrogen. The frozen cells werescraped and lysed in RIPA buffer. Insoluble material was removed bycentrifugation at 15,000×g for 15 min and the supernatant was collectedfor immunoprecipitation and immunoblotting. Phosphotyrosine-containingproteins from the clarified lysates were immunoprecipitated withmonoclonal anti-phosphotyrosine antibody and separated byelectrophoresis in 4–12% SDS-polyacrylamide gels under reducingconditions followed by electrotransfer to PVDF membrane andimmunoblotting with a polyclonal anti-phosphotyrosine antibody. Theblots were developed using the ECL chemiluminescence detection system(Amersham). Total protein content in the clarified cell lysates wasassayed using the Bradford method (Bradford, 1976).

Statistical Analysis. Where applicable results were expressed as themean±SEM and subjected to unpaired Student's t test. Within groupcomparisons were analyzed using one-way analysis of variance (ANOVA).p<0.05 was considered statistically significant.

Amylase release. CCK was a potent stimulus of amylase release. Maximumstimulation was seen at 10 nM (FIG. 14). Although glucagon (10 or 100nM) by itself had no effect on amylase release, when combined with CCKit inhibited, but did not fully abolish, CCK-induced amylase release(FIG. 15; n=20, p<0.01). GLP-1 and insulin, either alone or combinedwith CCK, did not influence amylase release (FIG. 15). We also examinedexendin-4 (concentrations ranging from 10 nM to 10 nM) for potentialeffects on amylase release, and, similar to GLP-1, it did not appear toinfluence amylase release. As GLP-1 and glucagon might be expected toraise cAMP levels in AR42J cells we looked at the effect of 8-Bromo-cAMP(8-Br-cAMP), a cAMP analog, on amylase release to look for specific cAMPeffects. While 8-Br-cAMP appeared to have no effect on amylase releasewhen given alone, it reduced CCK-induced amylase release (FIG. 16). Wealso used thapsigargin and ryanodine, specific inhibitors of ryanodinereceptors/ER Ca²⁺ release channels and of the ER Ca²⁺ pumps,respectively, alone and in combination with CCK, to investigate the roleof a rise of intracellular calcium on amylase release. The combinationof thapsigargin and ryanodine decreased, but did not fully inhibit,CCK-induced amylase release (FIG. 17; n=3, p<0.01). Naf, which mimicsCCK's effects on amylase release in acinar tissue (Vajanaphanich et al.,1995), did like-wise in the AR42J cells. Genestein (300 μM), thetyrosine kinase inhibitor, decreased CCK-mediated amylase release,especially at the early time points of the CCK treatment, whilevanadate, the tyrosine phosphatase inhibitor, increased significantlybasal and CCK-mediated amylase release (FIG. 18). We have shown thatwhen beta cells of the pancreas are treated with GLP-1 for 24 h there isan increase in glucose- and GLP-1-mediated insulin release (Wang et al.,Endocrinology, 1995). We therefore looked for any long-term effectsGLP-1 might have on amylase release. Pre-incubation of AR42J cells for8, 24, 48 or 72 h with GLP-1 (10 nM) and insulin (100 nM) did notincrease basal or CCK (1 nM)-induced amylase release.

[Ca2⁺]_(l) responses to CCK in AR42J cells. Under the presentexperimental conditions, most (85%; n=35) of the AR42J cells respondedto 1 nM CCK with a transient increase in [Ca²⁺]_(l). FIG. 6A shows arepresentative example of the CCK-induced [Ca²⁺]_(l) transients, whichcommenced after 5–25 seqs following exposure to CCK and peaked withinthe next 5–15 secs. The peak [Ca²⁺]_(l), assessed from the peak indo-1fluorescence ratio (IFR), exceeded the resting IFR by 2.5–3.5 fold.Relaxation of the [Ca²⁺]_(l) transients commenced immediately followingthe peak and usually consisted of an initial rapid phase, followed by aplateau and a slower final phase. After the [Ca²⁺]_(l) transient,baseline [Ca²⁺]_(l) decreased below the level of resting [Ca²⁺]_(l),measured prior to the exposure to CCK (FIG. 19A). During the subsequentrest, baseline[Ca²⁺]_(l) showed a gradual increase, but usually did notfully recover to the control levels within 10 min. [Ca ²⁺]_(l)transients elicited by a repeated exposure to CCK prior to a fullrecovery of resting [Ca²⁺]_(l) were reduced by 30–40% vs. the preceding[Ca²⁺]_(l).

The CCK-induced [Ca²⁺]_(l) transients were almost completely abolishedin cells pretreated with 10 μM ryanodine and 500 μM thapsigargin (FIG.19B; n=7). These results support the concept that in acinar cells, theER is the major source of changes in [Ca²⁺]_(l) induced by CCK (Muallemet al., 1988; Ochs et al., 1983). Consistent with this idea, exposuresto CCK added to a nominally Ca²⁺-free superfusing solution (FIG. 19C)did not appreciably affect the rate of rise or the magnitude of the[Ca²⁺]_(l) transients (n=5). However, as shown in FIG. 19C, a reductionin the extracellular Ca²⁺ shortened the duration of the [Ca²⁺]_(l)transients, suggesting, as shown before (Muallem et al., 1988); Ochs etal., 1983), that extracellular Ca²⁺ may play a role in sustaining thedelayed component of the [Ca²⁺]_(l) transient initiated by CCK-inducedER Ca²⁺ release.

[Ca²⁻]_(l) responses to GLP-1 in AR42J cells. Exposure to GLP-1 elicited[Ca²⁺]_(l) responses in approximately 50% (n=27) of AR42J cells. TheGLP-1-induced transients (FIG. 20A) displayed considerable variability,but usually developed at a slower rate and attained smaller amplitudes(1.5–2.5 fold increase over resting IFR) than the [Ca²⁺]_(l) responsesto CCK. Moreover, the GLP-1-induced [Ca²⁺]_(l) transients relaxed at aslower rate than those induced by CCK (FIGS. 20A vs. 19A and 20B,C).FIG. 20B shows the effects on [Ca²⁺]_(l) of CCK applied<10 min after anexposure to GLP-1 in the same cell. In experiments of this type, theCCK-induced transients retained their characteristic configuration (asin FIG. 19A) but reached smaller amplitudes. The latter effect can beattributed, at least in part, to a reduction of the [Ca²⁺]_(l) content,indicated by a reduction in the baseline IFR, and/or partial depletionof the ER Ca²⁺ content (see FIG. 19). On exposure to CCK for a secondtime the amplitudes were even smaller (FIG. 20C). Pretreatment withryanodine (100 μM) and thapsigargin (500 μM) virtually abolished[Ca²⁺]_(l) responses to GLP-1. Taken together, these results indicatethat CCK and GLP-1 have access to the same intracellular pools of Ca²⁺,presumably the ER, but perhaps release Ca²⁺ by differing mechanisms.Exendin-4, the GLP-1 homolog from Gila monster, had identical effects asGLP-1 on [Ca²⁺]_(l) but was approximately one order of magnitude morepotent. The GLP-1 antagonist, exendin 9–39 (Goke et al., 1993),inhibited GLP-1-induced calcium transients when used at a 10-fold higherconcentration than GLP-1.

Effects of glucagon and 8-bromo-cAMP on [Ca^(2−]) _(l) AR42J cells.Exposures to glucagon (10 nM) induced [Ca²⁺]_(l) responses in 70% (n=12)of AR42J cells. The [Ca²⁺]_(l) transients commenced briefly afterexposure to glucagon, developed at a relatively slow rate, peaked at200–250% of the resting IFR level and showed a prolonged, slowrelaxation (FIG. 21A). The [Ca²⁺]_(l) transients induced by CCK shortlyafter treatment with glucagon (or with both treatments addedsimultaneously) showed an attenuated rate of rise and a very slow rateof relaxation (FIG. 21B). Similarly, brief (60–300 sec) exposures to 0.1μM 8-bromo-cAMP, a membrane-permeable form of cAMP, usually did notmarkedly affect the rate of rise of the CCK-induced [Ca²⁺]_(l)transients but markedly slowed their rate of relaxation (FIG. 21C). Areduction in intracellular mobilization of [Ca²⁺]_(l) with theacetoxymethyl ester of dibutyryl cAMP in the presence of CCK haspreviously been shown in acinar cells (Kimura et al., 1996).

GLP-1 Binding. Specific ¹²⁵I-GLP-1 binding, as determined bydisplacement of total binding by the presence of 500 μM cold GLP-1, was0.64±0.16% (n=9, the amount of specific binding was significantlygreater than zero, p<0.01) of total radioactivity added and 27±3.2%(n=9) of total binding. Because of the low specific binding, a fullScatchard analysis was not performed.

cAMP levels. Intracellular cAMP levels were not altered in AR42J cellsby 1 h treatment with GLP-1 (0.1 to 100 nM) or IBMX (100 nM) in thepresence or absence of CCK (1 nM), or with CCK (0.1 to 100 nM) alone.While IBMX caused a slight increase in cAMP levels, in 3 experiments itwas not statistically different from non-IBMX-treated cells. Glucagon(10 nM) caused a 2-fold increase in cAMP levels in the presence andabsence of CCK (FIG. 22). Exendin-4 (0.1 to 10 nM) did not alter cAMPlevels.

RT-PCR of the GLP-1 Receptor. The presence of GLP-1 receptor mRNA wasdetected in AR42J cells by using RT-PCR. FIG. 23 shows that usingprimers identical to the known pancreatic GLP-1 receptor sequence(Thorens, 1992), PCR product of predicted size (bp928; see Egan et al.,1994) can be detected in AR42J cells and rat pancreas, but not in PCR ofwater control. The absence of any genomic DNA contamination isestablished as our primers span intronic sequences that would yield PCRbands of 1.8 K bases. No additional bands were observed corresponding tocontaminating genomic DNA PCR in our PCR reactions. The PCR reactionswere cloned, partially sequenced and identified to be the beta cellGLP-1 receptor.

Western blot analysis of GLP-1 expression. Using an antibody against theN-terminal region of the GLP-1 receptor, specific bands were obtained at65 and 45 kDa in the positive control cells, the RIN1046-38 cells, andin the AR42J cells. These have been shown to correspond to the matureand core-glycosylated GLP-1 receptors, respectively (FIG. 24).

Tyrosine Phosphorylation Studies. In the absence of any stimulation,some proteins exhibited a basal level of phosphorylation which wasincreased in the presence of CCK and NaF, but not GLP-1 (FIG. 25). Fourproteins (46, 66, 120 and 190 kDa) were the most obviously influenced inthe presence of CCK with at least a 2-fold increase in thephosphorylation levels of those proteins. Genistein decreased tyrosinephosphorylations induced by CCK and diminished CCK-mediated amylaserelease, as already shown in FIG. 18.

AR42J cells respond in a physiological manner to CCK as evidenced byinduction of amylase release in a concentration-dependent manner andincreased intracellular calcium. CCK also induced protein tyrosinephosphorylation as had previously been shown (Lutz et al., 1993). CCKinduced substantial increases in tyrosine phosphosubstrates of kDa 190,120, 66 and 46 on the basis of apparent molecular masses when separatedon SDS-polyacrylamide gels. Two of those phosphorylations, 120 and 66kDa, have already been described (Id.). Inhibition of tyrosinephosphorylation by genistein inhibited amylase release and alsodecreased tyrosine phosphorylation events. This suggests that in AR42Jcells, as in acinar cells, that tyrosine phosphorylation is involved inregulated amylase secretion. Insulin induced phosphorylation of mostprobably its own receptor beta subunit at 97 kDa. NaF, a well knownactivator of G proteins (Rivard et al., 1995), has previously been shownto mimic CCK's effects in acinar cells in that it increases amylaserelease and increases tyrosine kinase activity in acinar cells (Id.).NaF mimics CCK's effects on tyrosine phosphorylation events in AR42Jcells and therefore lends credence to the hypothesis that there exists afluoride-sensitive G protein that functions as a transducer between theCCK receptor and tyrosine phosphorylation (Id.).

GLP-1 clearly increased intracellular calcium but did not appear toincrease amylase release alone or with CCK in AR42J cells. No increasein cAMP was demonstrated in the presence of GLP-1 though it was obviouswith glucagon. Malhotra et al. (1992), using rat acinar cells, statedthat exendin-4, the Gila monster venom that is homologous to GLP-1,potentiated CCK-induced amylase release and increased cellular cAMP butdid not discuss GLP-1 effects. However, increased cAMP was not seenuntil 10⁻⁸ M exendin-4 was used, at which concentration exendin-4 may beinteracting through other receptors (Id.). Likewise the effect onpotentiating CCK-induced amylase release (from 12% of total amylasereleased by CCK alone vs. 16% with exendin-4 and CCK together) was seenwith 10⁻⁸ M exendin-4 and reached statistical significance only at the15 min time point (p<0.02) of a time course of exposure to CCK for 1hour. The methods may not be sensitive enough to pick up such a verysmall and time-specific effect of GLP-1 or exendin-4 if it wereoccurring, and, once again, the effect on secretion shown by Malhotra etal. may be due to interaction with other receptors. In beta cells of thepancreas exendin-4 increases cAMP and insulin secretion withconcentrations as low as 10⁻¹⁰ M concentrations (Goke et al., 1993).Alternatively, due to low receptor affinity, small, acute changes incAMP levels with GLP-1 may not have been detected.

The response of AR42J cells is similar to that seen in peripheral cells(liver, fat, and skeletal muscle), which do not show an increase in cAMPlevels either (Valderde And Villanueva-Penacarrillo, 1996). It appearsthat GLP-1 might be coupled to either a different G-protein subtype thanin beta cells or to other G-protein subtypes. The CCK receptor has beenshown to be coupled to G_(i) subtypes as well as G_(q) subtypes inacinar cells (Schnefel et al., 1990). In AR42J cells, GLP-1 may becoupled to at least a G_(i) subtype and possibly other G-protein alphasubunits. In 3T3-L1 adipocytes, in which GLP-1 increases lipid synthesisand glucose uptake, it has been shown that the GLP-1 receptor is mostlikely coupled to a G_(i) subtype (Montrose-Rafizadeh et al., J. Biol.Chem., 1997) and that in CHO cells which overexpress the GLP-1 receptorit is coupled to other alpha subunits (Montrose-Rafizadeh et al.,Diabetes, 1997).

Similar to CCK, the rise in intracellular calcium induced by GLP-1 wasfrom the endoplasmic reticulum. However, the pattern of the calciumgradients was not the same as with CCK, implying that the signaling tothe release of calcium by CCK was possibly different from that byglucagon and GLP-1. GLP-1 did not increase tyro sine phosphorylationevents. This demonstrates once again the importance of tyrosinephosphorylation for regulated amylase release. It also demonstrates thatpathways independent of an elevation of intracellular calcium areimportant for the secretion of amylase. This is further underscored bythe results obtained in the presence of thapsigargin and ryanodine.While they prevented any rise in intracellular calcium they reduced, butdid not completely prevent, CCK-induced amylase release. So a rise ofintracellular calcium is necessary for the full expression ofCCK-induced amylase release but of itself it is clearly not sufficientto induce amylase release in AR42J cells.

Any cell type may contain diverse beta subunits of the GTP-bindingproteins (von Weizsacker et al., 1992). This could mean that dependingon the subtype activated, i.e., G_(q) by CCK or GLP-1, G_(s) by glucagonor GLP-1, or G_(i) by both CCK and GLP-1, a different G_(βy) subunit maybe released. A specific G_(βy) might then be required for the tyrosinephosphorylation events observed in AR42J cells as already described formitogen-activated protein kinase activation (Hawes et al., 1995). Italso raises the possibility that if two different G_(βy) subunits arereleased by the action of one hormone they might have additive orantagonistic effects on various down-stream events.

GLP-1 receptors are present on AR42J cells. Their activation by GLP-1and exendin-4 leads to increased intracellular calcium, probably fromthe ER. Their activation, however, does not lead to an increase inamylase release and CCK-induced amylase release is not potentiated.

Example 5

As discussed in Example 4, GLP-1 receptors are present on AR42J cells,and acute treatment of AR42J cells with GLP-1 raises intracellularcalcium in the cells. Furthermore, previous studies showed that,although dexamethasone promoted AR42J cells to become acinar-like cells(Christophe, 1994), betacellulin and activin A converted approximately10% of AR42J cells into insulin-producing cells (Mashima et al., J.Clin. Invest. 1996). Similarly, after exposure to hepatocyte growthfactor (HGF, also known as heptocyte scatter factor (HSF)), about 3% ofAR42J cells were insulin positive; whereas exposure to HGF and activin Aresulted in about 10% insulin positive cells (Mashima et al.,Endocrinology, 1996). There was no mention in either of the abovestudies relating to either GLP-1 or exendin-4. Furthermore, either GLP-1or Exendin-4 can convert AR42J cells into insulin-producing cells in fargreater numbers than combined treatment with activin A and betacellulinor combined treatment with HGF and activin A. The mechanism of theeffect by GLP-1 or exendin-4 may involve, as a final step, activation ofthe ERK/MAPK pathway, as inhibition of ERK activation prevented theinsulin and glucagon production.

Materials. AR42J cells were obtained from ATCC (Rockville, Md.). GLP-1,exendin-4 and exendin 9–39 (the GLP-1 receptor antagonist) were fromBachem (Torrance, Calif.). Anti-insulin and anti-glucagon antibodieswere from Linco (Charles, Mo.). Anti-rat ERK1/2 antibody (ERK1-CT) andMyelin Basic Protein (MBP) were purchased from Upstate BiotechnologyIncorporated (Lake Placid, N.Y.). Insulin radioimmunoassay reagents werefrom Peninsula Laboratories (Belmont, Calif.). Protein measurementreagents were obtained from Bio-Rad (Hercules, Calif.). Peroxidase ABCkits were obtained from Vector Laboratories (Burlingame, Calif.). Tian™One Tube RT-PCR system was purchased from Boehringer Mannheim(Indianapolis. Ind.). Deoxyribonuclease I was obtained from Gibco BRL(Gaithersburg, Md.). Glass coverslips were from VWR Scientific(Baltimore, Md.). The protein kinase C (PKC) inhibitor1-o-Hexadecyl-2-o-methyl-rac-glycerol (PKI), and the MAP kinase kinase(MAPKK) inhibitor, PD98059, were from Calbiochem (San Diego, Calif.).

Cell culture. AR42J cells were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum (FBS), 100 IU/mlpenicillin, 100 μg/ml streptomycin and 2 mM glutamine. Cells frompassage 23–35 were used throughout this study. Cells were routinelyplated at a density of about 10⁵ cells/ml in 12-well cluster dishes oron coverslips and incubated in a humidified incubator at 37° C. with 95%air and 5% CO₂.

Immunocytochemistry analysis. Cells were cultured on glass coverslips,washed with phosphate buffered saline (PBS) to remove serum, and fixedwith 0.5% glutaraldehyde in PBS. Cells were permeabilized with 0.2%Triton X-100 for 5 min and the rest of the procedure was carried out atroom temperature in a humidified chamber. Suction was used to removereagents between each step but drying of specimens was avoided.Sufficient reagent was used to cover each specimen (approximately 1 or 2drops was usually adequate). The coverslips were incubated in 0.3% H₂O₂in PBS for 30 min to quench endogenous peroxidase activity and washed inPBS×3 times, followed by incubation with 2% goat serum in PBS for 30 minto block non-specific binding of IgG. Excess serum was removed byblotting. The specific primary polyclonal antisera (anti-insulin 1:300;anti-glucagon 1:300) were used. Antibody was diluted in PBS containing1% goat serum. This was applied to the coverslip and incubated at roomtemperature for 1 h. The coverslips were washed ×3 times in PBS, eachtime for 5 min, then incubated with biotinylated second antibody for 1 hand washed ×3 times with PBS. Avidin-biotin-peroxidase complex in PBSwas applied for 30 min. Immunoperoxidase labeling was performed with aVectostain ABC kit (Vector Labs, Burlingame, Calif.). After extensivewashing in PBS for 4–5 times (each 5 min), the coverslips were incubatedin diaminobenzidine tetrahydrochloride (DAB) in PBS, with 0.01% hydrogenperoxide for 3 min. The reaction was stopped by washing the coverslipsin PBS and examined under a light microscope. To confirm specificstaining, samples incubated with preabsorbed primary antibody were usedas negative control, and the insulin producing cell line RIN 1046-38cells were used as positive control for our experiments. Theavidin-biotin-peroxidase (ABC) procedure was performed according tomethods known in the art (Hsu et al., 1981).

Measurement of immunoreactive insulin. AR42J cells were cultured asbefore in 12-well cluster plates. When cells reached 60% confluence,they were treated with GLP-1 for 3 days. At the beginning of theexperiments, an aliquot of the medium was taken in order to assay theinsulin accumulation in the medium. Then the cells were twice washedwith Kreb's Ringer Balanced Buffer (KRBB) and incubated in the samebuffer containing 10 mM glucose for another 1 h. The medium wascollected and kept at −20° C. until insulin levels were assayed by RIA(See Example 1; Wang et al., Endocrinology, 1995). The cells were washedwith PBS and detached with 0.25% typsin and 0.02% EDTA. The cell pelletwas collected and lysed with formic acid for protein determination bythe Bradford method (Bradford, 1976), using bovine-globulin as standard.

Reverse-transcription polymerase chain reaction (RT-PCR). Total RNA wasisolated from treated AR42J cells by the methods of Chomczynski andSacchi (1987). The total RNA samples were pretreated with DNAse in 20 mMTris-HCL (pH 8.4), 2 mM MgCl₂ and 50 mM KCl to remove any traces ofcontaminating genomic DNA. RT-PCR was undertaken in a volume of 50 μl ofbuffer containing 50 mM KCl, 10 mM Tris-HCl, 3.5 mM MgCl₂, 200 μM eachdNTPs, 0.4 μM each of sense and antisense primers to rat insulin I andII (insulin sense primer=5′TGCCCAGGCTTTTGTCAAACAGCACCTT3′; insulinantisense primer=5′CTCCAGTGCCAAGGTCTGAA 3′). Amplification wasundertaken for 25 cycles at denaturing temperature 94° C. for 1 min,annealing temperature 60° C. for 45 sec and an extension temperature 72°C. for 1 min. mRNA from RIN 1046-38 cells were used as a positivecontrol. In the case of glucagon RT-PCR, the denaturing and extensiontemperature was similar to insulin except the annealing temperature was65° C. for 1 min (glucagon sense primer=5′GTGGCTGGATTGTTTGTAATGCTGCTG3′; antisense primer=5′CGGTTCCTCTTGGTGTTCATCAAC3′). The RT-PCR products were visualized byethidium bromide staining on 2% agarose gels.

MAP Kinase Activity. After treatment, 60 mm dishes of 80% confluentcells were lysed at 4° C. in lysis buffer (in mM): 50 TRIS-HCl, PH 8,150 NaCl, 5 EDTA, 1% NP-40, 0.25% sodium deoxycholate, 1 NaF, 10 sodiumpyrophosphate, 0.1 PMSF, 1 sodium orthovanadate, 20 μg/ml aprotinin, and10 μg/ml leupeptin. The cell lysate was clarified by centrifugation at16,000×g at 4° C. for 20 min. The clarified cell lysate wasimmunoprecipitated overnight at 4° C., rotating with 4.5 μg of ERK1-CTantibody and 40 μl of packed protein G+protein A agarose resin (OncogeneResearch Product, Cambridge, Mass.). The immune pellet was assayed forMAPK activity using MBP as the substrate. MBP (18.6 μg) wasphosphorylated at 20° C. for 10 min in a final volume of 60 μlcontaining 20 mM Hepes, PH 7.4, 10 mM MgCl₂, 1 mM DTT, 20 uM unlabeledATP and 40 μCi (3,000 Ci/mmol) [³²P]-ATP. The reaction was terminated bythe addition of 25 μl of 3× Laemmli sample buffer and heating at 70° C.for 10 min. MAPK activity was assessed by SDS-PAGE and auto-radiography.The autoradiograms were quantified by densitometry.

Amylase Assay. For amylase determination, cells were washed free ofmedium with 2 ml PBS. Incubation was then carried out in DMEM containing15 mM HEPES, 0.2% BSA and 0.01% soybean trypsin inhibitor. CCK (1 nM)was added for 50 min at 37° C. The incubation medium was thenimmediately removed for amylase determination and the cells were againwashed in 2 ml ice-cold PBS. Lysate buffer containing (in mM) 130Tris-HCl, 10 CaCl₂, 75 NaCl, and 0.2% Triton X-100 (pH 8.0) was added tothe cells and the lysates were then collected for total amylase (Ceskaet al., 1969). The released amylase was expressed as the percentage ofthe total amylase in the cells.

Statistics. All data values are shown as mean±SEM, and the differencesamong the treated groups were analyzed by one factor ANOVA analysis.Differences between treated and non-treated cells were analyzed usingthe Students' t test. p<0.05 was considered significant difference.

Effects of GLP-1 on the expression of insulin and glucagon. After GLP-1or exendin-4 treatment, AR42J cells convert to insulin-containing cells.Using anti-insulin antibody, intense immunostaining was present in AR42Jcells. In contrast, no immunostaining was observed in AR42J cells nottreated with GLP-1. Preabsorption of the antibodies with an excess ofinsulin and glucagon prevented staining (FIG. 26).

With 1 nM GLP-1, ˜10% converted into insulin-positive cells after 3days. When 10 nM GLP-1 or 0.1 nM exendin-4 were used for 3 days, ˜25% ofthe AR42J cells converted into insulin-positive cells. In some areas ofthe slides whole sheets of contiguous cells became positive for insulin.An occasional glucagon-positive cell appeared as early as 24 h. By 48 h,20% of all treated AR42J cells were glucagon positive, with ˜6% of thecells being insulin-positive. By 72 h fully half of all the treatedcells contained glucagon. The number of cells contained glucagondeclined thereafter but still ˜25% of cells remained insulin-positivefor at least 7 days (FIG. 27). The presence or absence of dexamethasonein the culture medium did not in any way influence the number of cellsthat converted to “endocrine” cells in the presence of GLP-1. WhenPD98059 (50 μM), a selective inhibitor of MEK which phosphorylates andactivates ERK, or PKI (300 μM) were added concurrently with GLP-1,conversion of the cells did not occur.

Insulin release. After a 3 day period of treating AR42J cells with 1 nMGLP-1, insulin was readily detected in the culture medium byradioimmunoassay. Over 3 separate cultures 5.1±0.4 pg insulin/μg protein(mean±SD) was present in the cell culture medium from the 60–72 h timeperiod. To investigate whether glucose could induce insulin secretionfrom 3 day-GLP-1-treated and non-treated cells, the medium was removedand the cells washed with glucose-free KRBB×3 times. This was followedby the addition of KRBB containing 10 mM glucose for 1 h and the cellsmaintained at 37° C. The incubating buffer was collected and insulinmeasured. Insulin was zero from the control cells, whereas there was0.65±0.15 pg insulin/μg protein present in the buffer of cells that hadpreviously seen 3 days of GLP-1. Insulin secretion was barely detectedin the presence of 200 μM PKI or in the presence of PD98059 (50 μM).

RT-PCR analysis. RT-PCR analysis demonstrated a 187 bp rat insulin I andII mRNA in GLP-1-treated AR42J cells for 3 days. The RIN cells were usedas positive control. In this experiment, RNA was pretreated with DNAse,only a mRNA fragment of insulin I and II with the predicted length wasamplified, thus the band that appeared at 187 bp was the specificinsulin mRNA product (FIG. 28A). In contrast, no RT-PCR products weredetected in the negative control or in non-GLP-1-treated cells. Northernblot analysis of GLP-1-stimulated AR42J cells was faintly positive andtherefore the band scanned poorly. Glucagon mRNA at 236 bp was detectedin GLP-1-treated AR42J cells at 48 h (FIG. 28B).

MAP kinase activity. ERK activation was readily detected in AR42J cells.Its activity was markedly increased with GLP-1 and exendin-4, the Gilamonster venom peptide, that is 52% homologous to GLP-1 and been shown tobe an insulin secretagogue (Goke et al., 1993). Exendin-4 was about100-fold more potent than GLP-1 (FIGS. 29A and 29B). PKI (300 μM) alonedecreased MAPK activity to less than that of control cells.

Amylase change. Incubating AR42J with dexamethasone for 72 h increasedamylase content 6.6-fold (12.57 U/l) in the cells compared withnon-dexamethasone-treated cells (1.88 U/l). When GLP-1 was addedtogether with dexamethasone, the total amylase content was decreasedcompared to dexamethasone treatment alone (7.76 U/1). The acute responseto CCK (1 nM) was also decreased in the cells that were pretreated withGLP-1 for 72 h (FIG. 30).

GLP-1 induces AR42J cells to differentiate into pancreatic endocrinecells, or, at the very least, into cells with endocrine traits. Inconjunction with this observation, the same pattern occurs in thedeveloping embryonal pancreas (Guz et al., 1995). Glucagon is the firsthormone detected (Rall et al., 1973). It is postulated that cellscontaining glucagon are precursor cells for various other types of isletendocrine cells, and that they, in turn, arose from ductal epithelium(Guz et al., 1995). But the mechanisms regulating formation anddifferentiation of the pancreatic hormone-producing cells is stilllargely undetermined. GLP-1 turns on glucagon production very early inAR42J cells and this is then closely followed by insulin production.Eventually the majority of the “endocrine” AR42J cells areinsulin-producing cells as the glucagon production wanes. Exendin-4 waseven more potent than GLP-1 as a factor for insulin production in AR42Jcells. Some insulin-containing cells were seen in the presence ofconcentrations as low as 10⁻¹¹ molar exendin-4. GLP-1 (and/or aGLP-1-like peptide, perhaps resembling exendin-4) may be adifferentiation factor in the embryo for islets. Such a peptide would beexpected to be present in high concentrations locally as the pancreas isforming from the primitive gut.

Glucagon has been hypothesized to be the signal for differentiation ofthe beta cells by increasing cAMP which would lead to a decrease in cellproliferation and to changes in macromolecular synthesis, culminating inthe beta cell phenotype. (Rall et al., 1973) This might still beapplicable in the AR42J cells. As glucagon is the hormone seen first inour system it could be the signal for insulin production. GLP-1 producedin the primitive gut might be the signal for the glucagon expression(and subsequently the insulin expression), which would lead to thefurther formation of endocrine cells and islet-like structures.

The final common pathway to “endocrine” cell differentiation in AR42Jcells, as well as other cell types, is likely through the ERK/MAPKpathway. GLP-1 or Exendin-4 produce little or no insulin staining inAR42J cells and no insulin into the medium when the ERK activity isinhibited. Little or no insulin in the presence of GLP-1 and a PKCinhibitor is observed. As the GLP-1 receptor is known to be G-proteinlinked, is present on AR42J cells, and raises intracellular calcium inAR42J cells (see Example 4), its activation by ligand binding probablyleads to PKC activation as well as other as yet undetermined down-streamevents (Nishizuka, 1984; Zamponi et al., 1997). PKC, in turn, has beenshown to be one of the factors that activates the MAPK pathway(Offermanns et al., 1993; Siddhanti et al., 1995). Therefore, blockingPKC activation by GLP-1 probably lead to diminishing MAPK activity andprevented the development of the “endocrine” cell phenotype.

Moreover, not all cells convert to “endocrine” cells with GLP-1, evenwith incubations as long as 7 days. The treated AR42J cells possess bothexocrine and neuroendocrine properties, as has been described foruntreated AR42J cells (see Christophe, 1994). Morphologically, variouspopulations of the treated cells do not appear the same. Thus,sub-populations of cells may be present in the untreated AR42J cells.Specifically, some of these populations may possess the GLP-1 receptor,and others may not. Cell preparations made from the total population ofAR42J cells possess GLP-1 receptors by Western blotting, PCR analysisand partial sequencing. On sequencing, the receptor is identical to thatfound on beta cells and which has been fully characterized already (seeExample 4). Furthermore, at least 50% of the AR42J cells increaseintracellular calcium in response to GLP-1. Therefore, GLP-1 probablyactivates a series of events which require increased intracellularcalcium and as yet other hitherto unknown factors which are definitelypresent in AR42J cells and commit them to become “endocrine” cells.

Example 6

Subjects diagnosed with Type 1 diabetes can be selected for treatmentwith GLP-1 or exendin-4. The treatment method of Gutniak et al., 1992can be modified so that GLP-1 is administered for at least twenty fourhours by cannula in the antecubital vein. The cannula can be connectedto an insulin infusion system pumping between 0.03 and 4.80nmoles/kg/min GLP-1. Blood glucose levels can be monitored regularly,using methods well known in the art, during administration of GLP-1 andfollowing the twenty-four hour period of GLP-1 administration. Aftertwenty four hours of GLP-1 infusion, the subject shows levels of bloodglucose that approach normal levels and has a reduced need for insulintherapy.

Alternatively, subjects with Type 1 diabetes can be treated withexendin-4 by a single subcutaneous injection or by daily repeatedsubcutaneous injections of 0.01 nmole/kg to 0.4 nmole/kg. Blood glucoselevels can be monitored regularly after administration of exendin-4. Theneed for insulin replacement therapy should decrease and blood glucoselevels should approach normal levels.

The preceding examples are intended to illustrate, but not limit, theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

By contacting cells with GLP-1 or Exendin-4 as described above, it isunderstood that both GLP-1 or Exendin-4, substantially homologoussequences, or fragments thereof could be used together.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entirety are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

REFERENCES

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1. An isolated population of insulin-producing cells obtained fromnon-insulin-producing cells by a process comprising contacting thenon-insulin-producing cells in vitro for at least twenty-four hours withan amount of a substance effective to induce insulin production, whereinthe substance is selected from the group consisting of a GLP-1 peptide,a GLP-1 peptide containing one or more conservative amino acidsubstitutions at positions other than positions 7, 10, 12, 13 and 15 ofGLP-1, and a fragment of any one of the preceding GLP-1 peptides, andwherein the GLP-1 peptide or fragment thereof has the ability todifferentiate non-insulin-producing cells into insulin-producing cells.2. The population of claim 1, wherein the non-insulin-producing cellscomprise pancreatic cells.
 3. The population of claim 1, wherein thenon-insulin-producing cells comprise pancreatic acinar cells.
 4. Thepopulation of claim 1, wherein the non-insulin-producing cells comprisestem cells.
 5. The population of claim 1, wherein thenon-insulin-producing cells comprise pancreatic stem cells.
 6. Thepopulation of claim 1, wherein the non-insulin-producing cells comprisemammalian cells.
 7. The population of claim 6, wherein the mammaliancells comprise human cells.
 8. The composition of claim 1, wherein thecontacting is at least 3 days.
 9. The composition of claim 1, whereinthe substance is a GLP-1 peptide.
 10. The composition of claim 1,wherein the substance is a GLP-1 peptide containing one or moreconservative amino acid substitutions at positions other than positions7, 10, 12, 13 and 15 of GLP-1.
 11. The composition of claim 1, whereinthe substance is the fragment of a GLP-1 peptide, or a GLP-1 peptidecontaining one or more conservative amino acid substitutions atpositions other than positions 7, 10, 12, 13 and 15 of GLP-1.
 12. Amethod for differentiating non-insulin-producing cells intoinsulin-producing cells, comprising contacting the non-insulin-producingcells in vitro for at least twenty four hours with an amount of asubstance effective to induce differentiation of non-insulin-producingcells into insulin-producing cells, wherein the substance is selectedfrom the group consisting of a GLP-1 peptide, a GLP-1 peptide containingone or more conservative amino acid substitutions at positions otherthan positions 7, 10, 12, 13 and 15 of GLP-1, and a fragment of any oneof the preceding GLP-1 peptides, and wherein the GLP-1 peptide orfragment thereof has the ability to differentiate non-insulin-producingcells into insulin-producing cells.
 13. The method of claim 12, whereinthe non-insulin-producing cells comprise pancreatic cells, pancreaticacinar cells, stem cells, pancreatic stem cells, or mammalian cells. 14.The method of claim 13, wherein the mammalian cells are human cells. 15.The method of claim 12, wherein the contacting is at least 3 days. 16.The method of claim 12, wherein the substance is a GLP-1 peptide. 17.The method of claims 12, wherein the substance is a GLP-1 peptidecontaining one or more conservative amino acid substitutions atpositions other than positions 7, 10, 12, 13 and 15 of GLP-1.
 18. Themethod of claims 12, wherein the substance is the fragment of a GLP-1peptide, or a GLP-1 peptide containing one or more conservative aminoacid substitutions at positions other than positions 7, 10, 12, 13 and15 of GLP-1.
 19. A method of enriching an isolated population of cellsfor insulin-producing cells, comprising contacting non-insulin-producingcells in vitro for at least twenty four hours with an amount of asubstance effective to induce differentiation of non-insulin-producingcells into insulin-producing cells, wherein the substance is selectedfrom the group consisting of a GLP-1 peptide, a GLP-1 peptide containingone or more conservative amino acid substitutions at positions otherthan positions 7, 10, 12, 13 and 15 of GLP-1, and a fragments of any oneof the preceding peptides, and wherein the peptide or fragments thereofhas the ability to differentiate non-insulin-producing cells intoinsulin-producing cells.
 20. A method of promoting pancreaticamylase-producing cells to produce insulin, comprising contacting thepancreatic amylase-producing cells in vitro for at least twenty-fourhours with an amount of a substance effective to induce insulinproduction, wherein the substance is selected from the group consistingof a GLP-1 peptide, a GLP-1 peptide containing one or more conservativeamino acid substitutions at positions other than positions 7, 10, 12, 13and 15 of GLP-1, and a fragment of any one of the preceding peptides,and wherein the GLP-1 peptide or fragment thereof has the ability todifferentiate non-insulin-producing cells into insulin-producing cells.21. An isolated population of insulin-producing cells obtained fromnon-insulin-producing cells by a process comprising contacting thenon-insulin-producing cells in vitro for at least twenty-four hours withan amount of a substance effective to induce insulin production, whereinthe substance is selected from the group consisting of an Exendin-4peptide, an Exendin-4 peptide containing one or more conservative aminoacid substitutions at positions other than positions 1, 4, 6, 7 and 9 ofExendin-4, and a fragment of any one of the preceding Exendin-4peptides, and wherein the Exendin-4 peptide or fragment thereof has theability to differentiate non-insulin-producing cells intoinsulin-producing cells.
 22. The population of claim 21, wherein thenon-insulin-producing cells comprise pancreatic cells.
 23. Thepopulation of claim 21, wherein the non-insulin-producing cells comprisepancreatic acinar cells.
 24. The population of claim 21, wherein thenon-insulin-producing cells comprise stem cells.
 25. The population ofclaim 21, wherein the non-insulin-producing cells comprise pancreaticstem cells.
 26. The population of claim 21, wherein thenon-insulin-producing cells comprise mammalian cells.
 27. The populationof claim 26, wherein the mammalian cells comprise human cells.
 28. Thecomposition of claim 21, wherein the contacting is at least 3 days. 29.The composition of claim 21, wherein the substance is exendin-4.
 30. Thecomposition of claim 21, wherein the substance is an Exendin-4 peptidecontaining one or more conservative amino acid substitutions atpositions other than positions 1, 4, 6, 7 and 9 of Exendin-4.
 31. Thecomposition of claim 21, wherein the substance is the fragment of anExendin-4 peptide, or an Exendin-4 peptide containing one or moreconservative amino acid substitutions at positions other than 1, 4, 6, 7and 9 of Exendin-4.
 32. A method for differentiatingnon-insulin-producing cells into insulin-producing cells, comprisingcontacting the non-insulin-producing cells in vitro for at least twentyfour hours with an amount of a substance effective to inducedifferentiation of non-insulin-producing cells into insulin-producingcells, wherein the substance is selected from the group consisting of anExendin-4 peptide, an Exendin-4 peptide containing one or moreconservative amino acid substitutions at positions other than positions1, 4, 6, 7 and 9 of Exendin-4, and a fragment of any one of thepreceding Exendin-4 peptides, and wherein the Exendin-4 peptide orfragment thereof has the ability to differentiate non-insulin-producingcells into insulin-producing cells.
 33. The method of claim 32, whereinthe non-insulin-producing cells comprise pancreatic cells, pancreaticacinar cells, stem cells, pancreatic stem cells, or mammalian cells. 34.The method of claim 33, wherein the mammalian cells are human cells. 35.The method of claim 32, wherein the contacting is at least 3 days. 36.The method of claim 32, wherein the substance is exendin-4.
 37. Themethod of claim 32, wherein the substance is an Exendin-4 peptidecontaining one or more conservative amino acid substitutions atpositions other than positions 1, 4, 6, 7 and 9 of Exendin-4.
 38. Themethod of claims 32, wherein the substance is the fragment of anExendin-4 peptide, or an Exendin-4 peptide containing one or moreconservative amino acid substitutions at positions other than 1, 4, 6, 7and 9 of Exendin-4.
 39. A method of promoting pancreaticamylase-producing cells to produce insulin, comprising contacting thepancreatic amylase-producing cells in vitro for at least twenty-fourhours with an amount of a substance effective to induce insulinproduction, wherein the substance is selected from the group consistingof an Exendin-4 peptide, an Exendin-4 peptide containing one or moreconservative amino acid substitutions at positions other than 1, 4, 6, 7and 9 of Exendin-4, and a fragment of any one of the preceding peptides,and wherein the Exendin-4 peptide or fragment thereof has the ability todifferentiate non-insulin-producing cells into insulin-producing cells.40. A method of enriching an isolated population of cells forinsulin-producing cells, comprising contacting non-insulin-producingcells in vitro for at least twenty-four hours with an amount of asubstance effective to induce differentiation of non-insulin-producingcells into insulin-producing cells, wherein the substance is selectedfrom the group consisting of an Exendin-4 peptide, an Exendin-4 peptidecontaining one or more conservative amino acid substitutions atpositions other than 1, 4, 6, 7 and 9 of Exendin-4, and a fragment ofany one of the preceding peptides, and wherein the peptide or fragmentthereof has the ability to differentiate non-insulin-producing cellsinto insulin-producing cells.